Application of FTIR spectroscopy for analysis of the quality of honey

Every kind of honey is a very precious natural product which is made by Mellifera bees species. The chemical composition of honey depends on its origin or mode of production. Honey consists essentially of different sugars, predominantly fructose and glucose. There are also non – sugar ingredients like proteins and amino acids, as well as some kind of enzymes, such as: invertase, amylase, glucose oxidase, catalase and phosphatase. The fact that honey is one of the oldest medicine known worldwide is remarkable. Scientists all over the world have been trying to improve analytical methods as well as to implement new ones in order to reaffirm the high quality of honey the benefits of which may be distracted or disturbed. There are many methods and popular analytical techniques, including as follows: mass spectroscopy and molecular spectroscopy (especially FTIR spectroscopy). The infrared spectroscopy technique is one of the most common analytical methods which are used to analyse honey nowadays. The main aim of the task was to use ATR-FTIR infrared spectroscopy to compare selected honey samples as well as typical sequences coming out from certain functional groups in the analysed samples.


Introduction
Honey is a natural product produced by bees of Apis mellifera species from nectar or honeydew. It has a distinctive sweet taste due to a high concentration of sugar i.e. glucose and fructose.
During storage in the vast majority of honeys, a spontaneous crystallization process which determines their consistency takes place [1]. On one hand, the solid phase of honey contains glucose, which is less soluble than fructose. Moreover, glucose can crystallize spontaneously at the room temperature and can be in the form of α-D-glucose, monohydrate and α-D-glucose and anhydrous β [2][3]. On the other hand, the liquid phase makes up other ingredients, and the most important is the aqueous solution of glucose. The crystallization process of honey continues until equilibrium, i.e. when glucose monohydrate crystals stop growing and do not dissolve [4]. It should be noted that many factors affect honey crystallization. The chemical composition of honey, primarily the type and concentration of sugars, and water content play an important role in this process. Honey during crystallization, in which the ratio of glucose to water (G/W) is less than 1.7, often tends to delaminate. Crystallization of honey accelerates due to the presence of crystallization germs (i.e. air bubbles, dust, pollen or previously crystallized honey) [5]. The optimum temperature for the crystallization process of honey ranges from 10 °C to 14 °C. Lupano in his works [6] showed that in honeys stored at 20 °C large crystals were formed with the melting temperature (T m ) ranging from 45 to 65 °C, while in honey stored in temp. -20 °C small crystals were formed with the T m ranging from 25 to 45 °C.
The crystal structure of honey also depends on the nectar origin. Rapeseed honey is characterized by small and irregular crystals. Crystals in buckwheat honey can be of a considerable size and relatively regular shapes [7]. At this point it is worth to emphasise that the crystallization phenomenon does not adversely affect the quality characteristics of honey [8].
An important tool for determining the crystalline structure of natural honey and its purity is ATR-FTIR IR spectroscopy [9]. This spectroscopy can be successfully applied for the classification of the honey type by replacing the long and timeconsuming pollen analysis. Analysis of honey with the infrared spectroscopy can be used to determine the crystalline structure as well as the purity [2]. It also enables quick determination of the quantitative and qualitative components which define the specific quality of natural honey. The spectra allow distinguishing honeydew honey, artificial, or nectar honey, and the use of specialized software gives the ability to detect even the varieties of the nectars' honey.
FTIR spectrums of natural honeys may significantly differ with some wavelength ranges. It also should be emphasised that very often different bands (in samples) overlap and the sample may not be sufficiently characterized, which can be due to the presence of substances like polysaccharides, water and many other pollutants resulting in a wide band in analytically important spectral ranges. The objective of this paper was to specify certain properties of the selected 5 types of honey using the ATR-FTIR analysis.

Materials and Methods
Honeys were purchased in the retail network of the city of Lublin. Five types were analyzed, including buckwheat, multifloral, sunflower, linden honey and honeydew with honeydew leaf. According to the declaration of the manufacturers on the labels, honeys were harvested both in the European Community (EC) and non-EC countries. Before the analysis, the samples were stored in a dark place at the room temperature. Honeys have been naturally crystallized to form on the surface of a thin liquid layer. Physical and chemical properties of honey were determined in mixed samples and presented in Table 1. Water and extract content in honeys was determined by measuring the refractive index by Abbe refractometer Carl Zeiss (Jena, Germany) [10]. PH, free acidity and electrical conductivity of honey were determined as described earlier [10] using pIONneer 65 Meter (Radiometer Analytical, Villeurbanne, CEDEX-France) with a combined pH electrode (E16M340) and a 4-pole conductivity cell (CDC 30T) with a built-in temperature sensor.

FTIR measurements
The measurements of ATR-FTIR background-corrected spectra were carried out in solvents using a HATR Ge through (45° cut, yielding 10 internal reflections) a crystal plate for liquids and were recorded with 670-IR spectrometer (Varian, USA). Typically, 25 scans were collected, Fouriertransformed, and averaged for each measurement. The IR absorption spectra at the resolution of one data point per 1 cm -1 were obtained in the region between 4000 and 400 cm -1 . The instrument was purged with argon for 40 min before and during the measurements. The Ge crystal was cleaned with ultra-pure organic solvents (Sigma-Aldrich Co.) All measurements were taken at the room temperature (T = 23 °C). The spectra was measured both in the mixed and non-mixed samples. All experiments were carried at 20 °C. Table  1 presents physicochemical properties of the analysed honeys. All evaluated honeys met the limits required by the national legislation [11]

FTIR spectroscopic studies
Only naturally obtained honey has been used to characterize the spectroscopic properties. In order to compare the samples FTIR spectroscopy was used as an efficient method. Typically, the characteristic differences in the FTIR spectral analysis for natural honeys were observed. It was probably related to the content of carboxylic acids in different types of honey resulting from the floral origin, geographic location and possible environmental pollution [2,[12][13][14][15][16][17]. Similarly as for alcohols, the O-H stretching vibration band in carboxylic acids is very broad and occurs in the field of 3300-2500 cm -1 [18][19][20][21][22] with the maximum at 3000 cm -1 . This is the same area as the stretching vibration region for carbon and aromatic C-H groups [23]. Therefore, carboxylic acids are characterized by an irregular character of absorption in 3300-2500 cm -1 with a wide range of the O-H stretching vibration band for C-H [12]. Strong hydrogen bonds present in dimeric carboxylic acids are the reason for the significant extension of the band stretching vibration of the OH group [24]. The stretching vibration band of the C=O carboxylic acids group occurs between 1760-1690 cm -1 [13,25]. The exact position of the band depends on whether the acid is saturated or unsaturated, dimerized or associated, etc. The C-H band can appear in 1320-1210 cm -1 , and the deformation band of the O-H group approx. in 1440-1395 cm -1 and 950-910 cm -1 [26]. However, the band under 1440-1395 cm -1 cannot be distinguished from C-H bending bands, which also occur in the same frequency region. In general: we observe O-H stretching in 3300-2500 cm -1 [27]/ C=O stretching in 1760-1690 cm -1 [26]/ C-O stretching in 1320-1210 cm -1 [28]/ O-H bending in 1440-1395 and 950-910 cm -1 [29][30]. To facilitate the characterization of bands and comparison of honeys selected for testing Figure 1 shows an ATR-FTIR spectrum of the tested honeys with major high bands. Table 2 shows the exact position of the bands together with the assignment of relevant vibration to specific functional groups. Table 3 presents a comparative position of the maximum vibration of multifloral and linden honey made from mixing and after thorough mixing of the selected samples for testing.  Table 1.
ATR-FTIR spectra of 5 samples of pure quality honey were obtained in the same conditions, as was described in the materials and methods section. The spectra of the selected honeys observed between 3800 and 700 cm -1 shows four typical absorption zones dominated by two water bands at 3339 cm -1 ((O-H) and 1649 cm -1 (OH deformation). In the same figure (Fig.  2, Table 2 and Table 3) a band at 2932 cm -1 was observed, which according to Anios et.
al. (2015) also corresponds to C-H stretching of carboxylic acids and NH 3 stretching band of free amino acids. The band from approximately 1500-750 cm -1 corresponds to the most sensitive absorption region of the major components of honey, particularly the most suitable region to quantify honey sugar (about 60-75 %) and organic acids. The contribution of sucrose, glucose, fructose, show characteristic bands in the region between spectral region located at 900-750 cm -1 is characteristic for the saccharide configuration.
Authors in many publications define the following spectral peaks as important to sugar characterization.
The To sum up the above considerations, distinct differences in the intensity of the bands which are first of all characteristic for vibration of the OH groups should be also emphasised. These changes inform clearly about varied water content in honey samples selected for testing. The greatest intensity of these bands with the maximum at 3340 cm -1 was in case of buckwheat honey, while the lowest for linden honey and sunflower honeys. The area of bands with the intense maximum at approximately 1055 cm -1 coming from stretching vibrations of both C-O in C-OH and C-C group in the carbohydrate structure also seems to be very interesting. It clearly indicates varied sugar composition of tested samples.
Fructose is the essential sugar in honey with the average value of 36% ranging from 30% to 50% in typical samples. Glucose is the second most representative monosaccharide in honey and varies from 16% to 34% with the mean value of 25%. Very significant changes were found also within the area of 900-750 cm -1 which confirms the variable saccharide configuration.
In addition, it is noteworthy to present the differences in the band with the maximum at approximately 1440, 2880, 1410 and 1250 and 1190 cm -1 . The above mentioned bands vary with regard to location and this greatly changes the crystals, which have been made in the test samples of honey. These effects will be considered in the subsequent preparation of the topic presented in this article using methods of both spectroscopic and thermographic measurement with the use of differential scanning calorimetry (DSC) and measurements of Raman spectra. Table 2. Location of maxima of absorption bands FTIR [12-13, 29, 31-34], together with the assignment of appropriate vibration for selected honeys: buckwheat, multifloral, sunflower, linden, and honeydew honey made in terms of spectral 3700-900 cm -1 .