INVESTIGATION OF HEALTH EFFECT OF FRUIT JUICE FROM LINGONBERRY ( VACCINIUM VITIS-IDEA ) VIA PHYSICOCHEMICAL METHODS

https://www.journal-imab-bg.org 1731 ABSTRACT: Objective: Antioxidants reduce cell damaging effects of free radicals, they also are used as medications to treat various forms of brain injury. Forest fruits as a lingonberry contain a lot of flavonoids and antioxidant supplements which contribute to the protection of different types of cancers and cardiovascular health problems. Purpose: The aim of this study is to explore the relations between natural fluorescence in juice from lingonberry and its total phenolic content and total antioxidant capacity in view of the usefulness of these compounds for human health and hygiene of food. Materials and Methods: For this reason, the juices from lingonberry from the region of Velingrad ånriched with lactulose will be investigated by using optical methods (colour measuring, fluorescence spectroscopy), rheological properties and sensor analysis. It may be an alternative method for quantifying the phenols and vitamins in juice from wild berries. Results: The dependences between chemical parameters, colour characteristics and the ratio between the intensity of emission and the intensity of excitation for exciting wavelength 275 nm have been found. The dependence between antioxidant activity and total phenolic content also exists. Excitation in the UV region is suitable for distinguishing the phenolic content and antioxidant compounds. The most suitable wavelengths found to be 265 nm and 275 nm. Lingonberry juices with lactulose exhibited nonNewtonian behaviour and Ostwald-de-Waele model are applied to calculate its rheological properties. Conclusions: The natural fluorescence of the lingonberry juices was correlated with their antioxidant properties, total phenolic content and lightness. From fluorescence spectra three groups of fluorescence components are determined – phenolic acids, tannins and flavonoids. The obtained results and discussion presented here give the direction for further studies and additional test for validation of the correlation between fluorescence intensity and phenolic compounds. In the future investigation, the fluorescence in the visible region will be used for determining the content of vitamins. The fluorescence spectroscopy is a promising technique for the rapid screening of lingonberry juice for antioxidant and phenolic content. The rheological behaviour of pasteurised juice from lingonberry showed a pseudoplastic behaviour.


INTRODUCTION:
Lingonberries (Vaccinium vitis-idaea) are kind of bushes or subshrubs of Pyrene family, which are distributed in wide regions of the Northern countries such as Finland, Sweden and Norway. Its fruits are used for consumption as juice, jam, wine or tea. There is a growing interest in the investigation of fruits because they contain bioactive compounds such as phenolic compounds [1][2][3], anthocyanins, omega-3 fatty acids, vitamins [4]. These compounds have nutritional and medical application -they stimulate the immune system, modulating hormone metabolism and possess antibacterial and antiviral action [5][6][7]. In the numerous studies, authors reported that the foods from lingonberry could reduce the incidence of cancer, cataracts, macular degeneration and cardiovascular disease [8][9]. There are many methods to determine the phenolic content and antioxidant activity of berries juice -the most frequently used are high performance liquid chromatography (HPLC), mass spectrometry and fluorimetry [10][11]. In literature, there are reports that fluorescence spectroscopy is used to screen the antioxidant contents in coffee and tomato paste [12][13].
The aim of this study is to explore the relations between natural fluorescence in juice from lingonberry and its total phenolic content and total antioxidant capacity in view of the usefulness of these compounds for human health and hygiene of food. Front face fluorescence spectroscopy is a quick, non time consuming method with high selectivity and

INVESTIGATION OF HEALTH EFFECT OF FRUIT JUICE FROM LINGONBERRY (VACCINIUM VITIS -IDEA) VIA PHYSICOCHEMICAL METHODS
sensitivity, without the use of chemical reagents. It may be an alternative method for quantifying the phenols and vitamins in juice from wild berries.

MATERIALS AND METHODS: Samples and technologies:
The investigation used wild fruits Vaccinium vitisidaea L. from harvest 2015 in the region of Velingrad. The juice from lingonberry ånriched with polyphenols is obtained by using the technological scheme, which is presented in Figure 1. The pasteurised juices with adding of lactulose are investigated. Fig. 1. Technological scheme for producing pressed juice FRAP reagent with 0.1 ml of investigated juice. The reaction time was 10 min at 37°C in darkness, and the absorbance was measured at 593 nm.
Fluorescence spectra measurements: The sources used to measure the fluorescence spectra are 245 nm, 265 nm, 275 nm and 295 nm light emitting diodes (LEDs). A fiber optic spectrometer (AvaSpec-2038, Avantes) with a sensitivity in the (200-1100) nm range and a resolution of about 8 nm was used to measure the fluorescence spectra. The lingonberry juices were placed in a cuvette 10 mm x 10 mm and illuminated by LEDs.
Colour measuring: Using a software package VISIONlite ColorCalc for spectrophotometer Helios Omega with a cuvette of a 10 mm length, the colour parameters in CIELab colorimetric system have been obtained. All measurements have been carried out at room temperature. Colour parameters a, b and brightness L of tested samples have been measured. Parameters such as chroma Cab and Hue angle h ab were defined as follows: Sensor analysis: Quantitative descriptive test for sensor profiling is used [14] for determining the sensor characteristics (colour, taste, aftertaste, smell). The corrective estimation is guaranteed from a commission from 12 trained assessors. The intensity of each sensor characteristics are written in the seven balls linear scale.

RESULTS:
The juice from lingonberry with different concentration of lactulose have been investigated for the total content of anthocyanins and polyphenolic compounds. The results are presented in Table 1. By using FRAP method, the data for antioxidant activity have been obtained (Table 1).

METHODS:
The content of polyphenols and anthocyanins 0.5 -1 ml fresh plant material was triturated with quartz sand and 2-5ml 70% methanol in a mortar, quantitatively transferred to a flask with a reflux refrigerator. It is extracted for 20 min at 70 0 C three times. The content of anthocyanin pigment concentration expressed as cyanidin-3-glucose equivalent, is calculated. The contents of biologically active substances such as anthocyanin and phenolic components in the juice samples were measured spectrophotometrically.
Antioxidant activity: Ferric reducing antioxidant power (FRAP) assay was used for determining the antioxidant activity. The reaction was started by mixing 3.0 ml The connection exists between investigated chemical parameters in Table 1 and the fluorescence peaks in the visible region. The fluorescence spectra for excitation wave-length 245 nm, 265 nm, 275 nm and 295 nm are presented in Figure 2.

Fig. 2. Fluorescence spectra for juices from lingonberry
The rheology properties of the juice are investigated by using Ostwald-de Waele model. The results for the consistency index (K) and the flow behaviour index (n) are obtained in Table 2.
Colour characteristics in CIE Lab colorimetric system have been measured ( Table 3). The dependencies between chemical parameters, colour characteristics and the ratio between the intensity of emission and intensity of ex-citation for exciting wavelength 275 nm have been found. The dependence between antioxidant activity and total phenolic content also exists. The existing dependencies and its correlation coefficients are presented in Table 4.

DISCUSSION:
The research is focused on juices of wild berries and more specifically on cranberry juice. We have searched for relations between chemical compounds having importance The results from sensor analysis have been presented in Figure 3, where samples 1, 2 and 3 correspond to juices with 0.5 %, 1% and 1.5% concentration of lactulose.  The content of the polyphenol acids in cranberry juice is of high significance because they stimulate the proper functioning of digestive system helping the rational use of food, improve appetite, decrease the intake of excessive quantities of water, thus exercising a beneficial effect in the case of disorders of the digestive system. The contents of tannin in these fruits makes them have an antiinflammatory effect and an excellent antidiarrhetic, especially in some cases of catarrhal colitis. The role of cranberry for the proper flow of vital processes due to the vitamins, phytoncides organic acids, vegetable hormones, carbohydrates, pectin and aroma substances, enzymes, causing antioxidant activity of juices and their fluorescence. There is a variety of data in medical science that the presence of the above compounds in cranberry juice is the cause of its tonic, refreshing effect which increases human activity.
It is evident from Figure 2 that the emission intensity has the lowest value for excitation wavelength 245 nm. The fluorescence spectra are clearly distinguishable for 275 nm, and the ratio between excitation and emission intensities is the best for a 265 nm excitation wavelength. The pointed wavelengths are appropriate for determining the fluorescence components in juice from lingonberry. Phenolic acids have the adbsorption maxima in the 240 nm-290 nm range, and the excitation/emission intervals are observed as follows: 1. 245 nm/445 nm -it is connected with the chlorogenic acid in juice with 0.5% and 1.5 % lactulose. The similar results have been obtained in [14] from Mazina et al. for the apple juice.
2. 265 nm/426 nm -this is maximum for 1.5 % lactulose in juice from lingonberry, and this maximum is belonged to caffeic acids [15].
3. (230nm-315) nm/(345-405) in literature connected with tannins acids. This maximum is observed for all of the concentration of lactulose in the investigated juice.
The proposed method for obtaining the fluorescence spectra is very simple , developed compactly and cost effective, because the system uses LED light sources, which are cheaper than lasers and they can cover the range from deep UV to visible light. In the present study, the excitation wavelength in the UV range is used because the goal is to find qualitatively the phenols and anthocyanins. The excitation wavelength in the visible region will be used in the future investigation for qualitative determination for vitamins because they are also important for human health.
It is evident that the antioxidant activity decreases with raising the concentration of lactulose. Juices from lingonberry contain flavonoids, low molecular weight polyphenols, anthocyanins, which have an anticarcinogenic effect, anti allergic, antiviral and anticancer activity [17]. They have a greater content of antioxidants than grapes at about 12% [18]. Antioxidant activity of juices from lingonberry is due to the polyphenolic contents. These components help in the prevention of cardiovascular diseases, in treatments for Neurodegenerative diseases and amylotrophic lateral sclerosis [19]. The total phenolic content of juice from lingonberry with 0.5 % lactulose is two times lower than the juice from aronia melanocarpa [18].
The fluorescence spectroscopy is an advanced technique for the rapid screening of lingonberry juices for total phenolic contents. It is found that the dependence between ratio intensity of fluorescence and intensity of excitation for wavelength 275 nm and total phenolic content exist. A positive correlation was obtained between phenols and antioxidant activity. The similar relation is in accordance with literature data for apple juice [19].
The investigated juices have low brightness in the interval (20-22) a.u. with dominant red and yellow colors and with sharp Hue angle less than 15 0 . There is a positive correlation between brightness and the ratio of intensities of emission and excitation for wavelength 275 nm. Adding of lactulose slightly changed the brightness of the juices.
The taste profile is developed of fruit juices from lingonberry with lactulose, and the sensor analysis is made on the basis of the main indicators. The appearances and cranberry flavor are highlighted in the first sample. The low concentration of lactulose in juice is acceptable for consumers, and the juice is close to natural.
The rheological properties are very important for hygiene of food because they are changed dramatically [20] for different concentration levels during processing of fruit juices. In Figure 4 were shown flow and viscosity curves of pasteurized lingonberry juice. In this case, rheograms of viscosity versus shear rate show concave curves. This fact shows that lingonberry juices with lactulose exhibited non-Newtonian behavior and Ostwald-de-Waele model are applied to calculate its rheological properties. All the values of the flow behavior index are below 1 supporting the pseudoplastic behaviour of pasteurized juice ( Table 3). The same results have been reported from Kobus et all. for juice from carrots [21]. The index of consistency is greater for the juice with 0.5 % lactulose and index of flow behavior.

CONCLUSION
The natural fluorescence of the lingonberry juices was correlated with their antioxidant properties, total phenolic content and lightness. From fluorescence spectra three groups of fluorescence components are determined -phenolic acids, tannins and flavonoids. The obtained results and discussion presented here give the direction for further studies and an additional test for validation of the correlation between fluorescence intensity and phenolic compounds. In the future investigation, the fluorescence in the visible region will be used for determining the content of vitamins. It can be concluded that the fluorescence spectroscopy is a promising technique for the rapid screening of lingonberry juice for antioxidant and phenolic content.
The rheological behaviour of pasteurized juice from lingonberry showed a pseudoplastic behaviour. The pasteurization process can change not only the viscosity but also the rheological properties of lingonberry juice.