Investigation of the conditions for the formation of                                                   5-Hydroxymethylfurfurol in the production of honey wines and                               sea-buckthorn wine drinks


Evgeny Rozhnov1, Alexey Kazarskikh1, Marina Shkolnikova1, Lyudmila Tretyak2, Vladimir Voytsekhovskiy3, Nikolai Maksimiuk4, Mars Khayrullin5, Maksim Rebezov5,6,7*,  Zhanibek Yessimbekov8

1Biysk Technological Institute (Branch) of the Altay State Technical University, Biysk, Russia

2Orenburg State University, Orenburg, Russia

3National University of Life and Environmental Sciences of Ukraine, Kiev, Ukraine

4Yaroslav-the-Wise Novgorod State University, Veliky Novgorod, Russia

5K. G. Razumovsky Moscow State University of technologies and management (the First Cossack University), Moscow, Russia

6Ural State Agrarian University, Yekaterinburg, Russia

7Orel State University named after I.S. Turgenev, Orel, Russia

8Shakarim State University, Semey City, Kazakhstan

*Corresponding Author E-mail:



The article presents experimental data on some mechanisms for the formation of 5-hydroxymethylfurfural (HMF) in the production of sea-buckthorn wines and wine drinks with the addition of honey. Conducted research is necessary because, despite the unconditionally useful properties of honey, it is possible that it has a high content of such chemicals as heavy metals, alkaloids (even in minimal quantities), as well as HMF, the unwanted effect of the latter on the body is mutagenic, genotoxic, organotoxic effects, accompanied by inhibition of a number of metabolic enzymatic processes in the living cell, in addition, the concentration of HMF is used as a parameter characterizing the freshness of honey. In this regard, the Codex Alimentarius Commission has set a maximum limit for HMF in honey - 40mg/kg (80 mg/kg for honey originating from tropical regions), ensuring that honey is not subjected to intense heating during processing and is safe for consumption. Thus, the identification of the kinetic patterns of formation of HMF will allow establishing the optimal parameters for the production of high-quality and safe honey wines and sea-buckthorn wines. It is shown that when preparing this group of drinks, the heating temperature of the product (at the stages of preparing the wort, pasteurizing the finished drink, etc.) should not exceed 60°C with a total heating time of not more than 6 hours and the content of total titrated acids in the drink not more than 7g/dm3. The research has scientific and practical significance for beverage producers who use natural honey and vegetable raw materials with high acidity in production, as well as for the regions of Russia engaged in industrial production of honey, since the creation of scientifically based technologies and modes of its storage and processing will improve quality and safety of food produced on its basis.


KEYWORDS: honey, sea buckthorn, wine, 5-hydroxymethylfurfural (HMF), kinetics, Maillard reaction.





Honey is a natural product of the life of a bee and consists of 15–20% of water and 80–85% of dry substances, which are a mixture of carbohydrates (mainly glucose (28.0–36.8%) and fructose (36.6– 39.5%), as well as sucrose (up to 0.5%), trehalose (0.51–0.78%), isomaltose (0.55–1.60%), maltose (3.2–7.1 %) and other sugars)[1]. Honey contains a wide range of nitrogenous compounds, including up to 20 free amino acids (including aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, glycine, leucine, histidine, hydroxyproline, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and ornithine)[2] and protein compounds, as well as enzymes, a number of aromatic compounds, flavonoids, minerals, vitamins, etc.[3,4]. The composition of honey is not constant and depends on the sources of its collection, geographic territories and the entomology of bees[5,6,7]. In addition, external factors, such as thermal effects and storage conditions, have a great influence on the composition of honey collected and prepared for storage, as well as the products of its processing[8,9,10]. Despite its high beneficial properties, the high content of chemicals such as heavy metals, alkaloids (even in minimal quantities), as well as 5-hydroxymethylfurfural (HMF) make honey and its products practically unsuitable for human consumption[11,12]. Therefore, the Codex Alimentarius Commission set the maximum limit for HMF honey at 40 mg/kg (with a higher limit of 80 mg/kg for honey originating from tropical regions) to ensure that the product is not subjected to intense heat during processing and is safe for consumption[13]. The International Federation of Juice Producers (IFFJP) recommends a HMF concentration limit of 5–10 mg/dm3 in fruit juices and 25 mg/dm3 in concentrated juices[14]. In wines, the HMF content ranges from 2 to 25 mg/dm3, furfural 0.1–10 mg/dm3, and methylfurfural to 1 mg/dm3 [15]. The presence of simple sugars and many organic acids in honey (in particular, gluconic, 4-dimethylaminobenzoic, caffeic, para-coumaric, gallic, vanillic, lilac and chlorogenic acids)[16], as well as minerals, increases the formation of this substance[17].


According to the chemical structure, HMF is a cyclic aldehyde formed in food as a result of the degradation of carbohydrates in an acidic environment when heated during technological processing or during long-term storage[18]. The formation of HMF is a component of the process of melanoid formation (Maillard reaction)[19] between sugars and amino acids (Figure 1).


Fig 1: Melanoid formation reaction scheme[20]


The harmful effects of HMF on the human body are mutagenic, genotoxic, organotoxic effects, accompanied by inhibition of a number of metabolic enzymatic processes in a living cell[21]. The concentration of HMF is traditionally used as a parameter characterizing the freshness of honey (in fresh honey, the HMF content is insignificant and usually does not exceed 2–5 mg/dm3), since during storage and processing accompanied by heating it increases significantly. Thus, high concentrations of HMF indicate high storage or processing temperatures[22,23,24].

The use of honey in food allows giving the finished products not only sweet taste, but rounding it, giving the taste sense of sweetness greater harmony, as well as forming the so-called fullness of taste. In addition, the aromatic components of honey, represented by a wide range of volatile compounds, improve the aroma of ready foods. For example, the use of honey as a source of carbohydrates in the production of sea buckthorn wines and wine beverages allows improving the organoleptic properties of the drink, in particular, the characteristic aroma of sea buckthorn is transformed, a bouquet of subtle floral-caramel tones appear even with a slight use of honey in sweetening. Thus, taking into consideration the high reactivity of honey carbohydrates to the formation of HMF in acidic media substantial interest presents the study of the conditions of formation of this compound in the production of honey wine and sea buckthorn wine drinks, which are known to contain a sufficient amount of organic acids[25], which act as a catalyst in the reaction of non-enzymatic cinnamon (Maillard reaction), one of the products of which is HMF. It is known that during fermentation the amount of furan aldehydes practically does not change and they contain up to 5 mg/dm3 in dry wines, in liqueur - up to 25 mg/dm3 [26]. It is known that the presence of HMF in wort obtained from polysaccharide hydrolysates can substantially inhibit alcohol fermentation[27]. Therefore, the introduction to the practice the quality control of honey wort before fermentation and clarification of the modes of heat treatment of wort is very important.


Thus, the purpose of this study was to identify the kinetic patterns of the formation of HMF in obtaining honey wines and wine drinks from sea buckthorn under controlled conditions for heating the wort from sea buckthorn of five varieties grown in the Altai region of Russia.



The objects of research were model solutions of honey (initial concentration of HMF in the initial product - 1.14 mg/dm3) containing 200 g/dm3 reducing substances and 0, 5, 7 and 9 g/dm3 malic acid, as well as prototypes of sea buckthorn varieties "Altai", "Chui", "Inya", "Essel" and "Augustine" (harvest 2017) obtained by diluting with pure water to a content of titrated acids of 5 and 7 g/dm3, followed by sugaring with honey to a content of reducing carbohydrates 200g/dm3. Quantitative determination of reducing sugars was performed by a chemical method using potassium permanganate[28]. The content of titrated acids was determined by potentiometric titration in terms of the predominant malic acid[29]. The content of individual organic acids in wort samples was determined by capillary electrophoresis (Kapel 105M, Russia)[30]. To determine the amount of α-amino acids in model solutions of honey and prototypes of sea buckthorn wort, a colorimetric method based on color reaction with ninhydrin was used[31].


Model solutions of honey and prototypes of sea buckthorn wort were heated to a temperature of 60, 70 and 80°C and kept for 12 hours. Every hour, HMF content was measured spectrophotometrically (Shimadzu UV-1800, Japan) using a modified Winkler method using 4-aminoantipyrine and barbituric acid at pH 3.5 and detection of optical density at a wavelength of 550 nm (optical path length 10 mm)[15,32]. Unlike the classical Winkler method, in which the color of the solution remains unchanged for an insignificant time, the used modified Winkler method allows using these reagents to maintain a stable red-violet color for 15–30 minutes.



To study the kinetics of HMF formation in prototypes of sea buckthorn wort with the addition of honey, it was necessary to study the composition of organic acids used in the work of sea buckthorn varieties. Table 1 presents the results of determining the active acidity, total titrated acidity and mass concentrations of individual organic acids in samples of sea buckthorn wine materials obtained by capillary zone electrophoresis. A typical electrophoregram is shown in Figure 2.



Fig 2: Electropherogram of “Altai” grade sea buckthorn blood


Table 1 Different grade of sea buckthorn’s organic acid composition


Active acidity, рН

Identified organic acids (mg/dm3)

Titratable acidity, on conversion to malic acid, g/dm3








































The results presented in Table 1 show that all the studied sea buckthorn varieties predominantly contain malic acid (66.5–79.6% of the amount of identified organic acids). Thus, it is advisable to simulate the HMF accumulation process in honey wort using malic acid as a catalyst for the degradation of sugars when heated.


Model solutions containing honey and malic acid, before heating, were examined for the content of nitrogenous substances and α-amino acids, giving staining with ninhydrin. As the results of the study showed, the content of ninhydrin-reacting nitrogenous substances and amino acids in model solutions is 78.2±6.2 mg/dm3. Thus, it is possible to assert with sufficient confidence that the reaction of HMF formation is possible in model solutions of honey.


Temperature control of model samples of honey solution was performed using a water thermostat. In order to prevent evaporation of the liquid when heated, the experiment was carried out in flasks equipped with reflux condensers. The results of determining the accumulation of HMF in the experimental honey solutions are presented in Figure 3. The data obtained by heating honey solutions at 60°C for 12 hours are quite interesting. Experimental data showed that the maximum allowable amount of HMF under these conditions was not reached and was only 14, 5 mg/dm3, however, the overall increase in the HMF content compared to the control was 1.8 times. At the same time, as the temperature rises to 80°C, the accumulation of HMF increases by more than 4.2 times. Apparently, this is due to the reaction mechanism and an increase in the heating temperature of the solutions above 70°C leads to overcoming the energy barrier that prevents the formation of Schiff bases and their subsequent transformation into HMF.




In general, the kinetics of the formation of HMF in model mixtures and control experiment obeys an exponential law and is described by an equation of the form: ,, the kinetic curves are appropriate, and their linearization is achieved in semi-logarithmic coordinates ln[C(HMF)] – t.. The calculated coefficients k1 and k2 are presented in table 2.


Table 2 Kinetic equations of HMF accumulation in the honey model mixtures at different temperatures 

Heating temperature of the model mixtures. °С

Concentration of malic acid in the model mixture. g/dm3





0 (control)

















0 (control)

















0 (control)

















The next stage of research was to determine the conditions for the accumulation of HMF in experimental solutions of honey wort prepared on the basis of sea buckthorn juices. Sea buckthorn juice from the studied sea buckthorn varieties were obtained by direct pressing. Then they made dilution of juices with distilled water and honey sweetening to similar model values, however, it was decided to abandon the titrated acidity value of 9 g/dm3, since our studies previously established that this acidity value does not allow preparing a drink with high organoleptic properties. The 10 prototypes of the wort with honey and sea-buckthorn juice thus obtained were heated at 60°C for 12 hours. The results obtained from studying the HMF accumulation process in the test samples are presented in Figure 4.


Fig 3: Kinetics of 5-hydroxymethylfurfurol formation in the model mixtures at different temperatures and various malic acid concentrations


test a – concentration of titratable acids 5.0 g/dm3; test b - concentration of titratable acids 7.0 g/dm3

1 – Altaiskaya; 2 – Chuyskaya; 3 – Inya; 4 – Essel; 5 – Avgustina

Fig 4: HMF formation dynamics in the samples of wort with honey and sea buckthorn


It can be seen that the accumulation of HMF in the test samples of the wort has the same nature of the curves as in the simulation of this process with the use of solutions of honey. As expected, in test samples, the accumulation of HMF is faster, which is associated with a high content of nitrogenous substances through the use of sea buckthorn juice (the content of ninhydrin-reacting nitrogenous substances is on average 5 times higher than honey solutions). Thus, when heated to a temperature of 60°C, the maximum possible duration of heating at the titrated acidity of the wort 5.0g/dm 3 should be 9 hours, and at an acidity of 7.0g/dm3, not more than 6.



The results obtained during the research reveal some mechanisms for the formation of HMF in the production of sea buckthorn wines and wine beverages with the addition of honey. It is shown that when preparing this group of drinks, the heating temperature of the product (at the stages of preparing the wort, pasteurizing the finished drink, etc.) should not exceed 60°C with a total heating time of not more than 6 hours and the content of total titrated acids in the drink not more than 7g/dm3.

The experimental data obtained can be useful to producers of soft and alcoholic beverages using natural honey and vegetable raw materials with high acidity in production. The conducted studies are also of high scientific and practical importance for the regions of Russia engaged in the industrial production of honey, since the creation of scientifically based technologies and modes of its storage and processing will improve the quality and safety of food produced on its basis.


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Received on 26.02.2019           Modified on 18.03.2019

Accepted on 24.04.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(7):3501-3506.

DOI: 10.5958/0974-360X.2019.00595.X