Abstract
Introduction
Black mulberry (
For the food industry needs both fruits and leaves are important. Products of blackberry fruits processing are jams, juices, syrups, wine, distillates or canned goods [10]. However, all plant parts of
Shoots possess antirheumatic properties, they are used as analgesic agent and for reduction of blood pressure. The bark anesthetic action and used for pain relief in case of toothache and in the traditional medicine the bark extract reveal antiparasitic effect [15].
The aim of this study was to evaluate morphological characteristic of selected genotypes of black mulberry fruits grown in Slovakia, as well as biochemical, technological and antioxidant properties of different products prepared from the black mulberry fruits.
Materials and methods
Biological material
Black mulberry (
Morphological analysis of black mulberry fruits
The following properties were measured by morphological analysis: Fruit length in mm, Fruit width in mm, Fruit weight in mg,
Chemicals
All the chemicals used were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Centralchem (Slovakia).
Preparation of black mulberry products
The tested fresh fruits and food products were made by traditional procedure using the processing methods of native inhabitants of village Pukanec in Slovakia. Fresh matured fruits were randomly selected from the obtained fruits. Natural juice was obtained from fresh fruits without thermal treatment. For this processing, the homogeneously ripened fruits were selected and mixed with a blender. A sieve was used to eliminate the seeds (Fig. 1c), the extract was then pressed softly in order to increase the yield. Natural juice was obtained from fresh fruits using hot steam. Fresh fruits were conserved in honey with a ratio of 1 : 3 without sterilization. Fresh fruits were conserved in honey with a ratio of 1 : 3 with subsequent sterilization at 85°C. Jam was produced from fresh fruits by traditional procedure (using sucrose with ratio 1:5). Jelly was produced from fresh fruits by traditional procedure (using sucrose ratio 1:6). Syrup was produced from fresh fruits by traditional procedure with addition of sucrose with a ratio of 1 : 8. Liqueur was made from fresh fruits by traditional procedure with 90% ethanol (final concentration of ethanol in liqueur was 22%). Compote was made from fresh fruits with sucrose with a ratio of 1 : 2. Wine was made from fermented fresh fruits and production time was 21 days until the concentration of 12% ethanol was reached. Briefly, the black mulberry juice was put in 10-L fermentation tanks at 20°C with a stable atmosphere in dark room. Fermentation method consisted of the free fermentation produced by the native yeast present in the black mulberry juice.
From each of the products three samples were tested.
Free radical scavenging activity
Free radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to Brand-Williams et al. (1995) [16]. An amount of 0.1 mL of sample of juice, wine, syrup, liqueur; sample of fruits conserved by honey, jam, compote and jelly was homogenized in a mortar and 1 g of the blended mass was extracted for 5 min in 100 ml of distilled water, filtrated and after filtration the extract was used for measuring. For detection of free radical scavenging activity, the extract was mixed with 3.9 mL of DPPH radical (0.025 g was soluble in 96% ethanol and diluted as needed). Absorbance was registered at 515 nm in regular time intervals until the reaction equilibrium was reached (10 minutes) by using spectrophotometer (Genesys 20 UV-VIS, USA). The DPPH scavenging activity (% inhibition) was calculated to fresh matter (FM) by using of following equation:
Total anthocyanin content
Total anthocyanin content was determined by the pH-differential method as described Wrolstad, (1993) [17]. For preparation of extract, an amount of 1 g of sample was extracted with 40 mL of solvent ethanol: 0.1 M HCl –85 : 15%, v:v, centrifuged and supernatant was used for measuring. The extract of sample were diluted with buffer and the absorbance was read at 520 and 700 nm with spectrophotometer (Jasco V-560 UV/VIS, Japan). Content of anthocyanin was calculated in mg–1 per dm–3 of fresh matter (FM) as pelargonidin-3-glucoside with molar absorption coefficient of 22400 M–1 cm–1 and molecular weight of 433.2 Da.
Detection of pH values
The detection of the pH value of samples was carried out by pH meter (Mettler Toledo FE20K, Germany)
Content of reducing sugar
Reducing sugar content was determined spectrophotometrically at 540 nm by the method using 3,5-dinitrosalicylic acid [18]. Reducing sugar content was calculated as glucose equivalent in mg–1 per kg–1 of fresh matter.
Content of ascorbic acid
Ascorbic acid content was determined using 5% metaphosphoric acid (MPA) as the extracting solution according to the method of Campos et al. [19] with modifications. Content of ascorbic acid was expressed in mg–1 per 100 g–1 of fresh matter.
Statistical analysis
Experimental data on fruit traits were evaluated by basic statistical variability indicators using the Microsofttrademark Excel® program. Dependency rate between the tested traits was expressed using the linear correlation analysis exploiting the relevant software [20].
Results and discussion
Morphological characterization of fruits
The medium fruit weight ranged from 1.42 g (MN-14) to 7.26 g (MN-1). Results on distinct differences between the traits of tested genotypes are shown in Table 1. Variation coefficients indicate medium (10.55-MN-17) and very high degree (36.22 –MN-8) of variability for the trait. Variability of fruits size is strongly affected by the genotype but in the same time should be taken into account the conditions of maturation. Black mulberry fruits are maturing gradually (Fig. 1a), therefore the fruits are picked up in a time period of 2, even 4 weeks and this could affect the variability of size of berries (Fig. 1b).
Medium fruits length ranged from 13.51 mm (MN-14) to 29.20 mm (MN-12). The variation coefficients referred from low (5.76% with MN-17) to high degree of variability (25.67% with MN-45). Medium fruits width ranged from 11.88 mm (MN-14) to 21.12 mm (MN-2). The variation coefficient showed the values from low degree (3.59% for MN-27) up to high degree of variability (24.39% for MN-8). Significant differences among the genotypes of all tested fruit traits were observed (
Black mulberry fruit size usually is characterized by the weight, height and width. The results of the present study (1.42–7.26 g) differ from the previous experiments in which the weight reported was from 2.96 to 6 g [21, 22]. However, the length and width of fruits in our report (from 13.51 to 29.20 mm and from 11.88 to 21.12 mm, respectively) correspond to reports of other authors with the the length accounting 14–35 mm and width 13–22 mm [23, 24].
Linear correlation between the evaluated traits indicates very strong positive correlation between the fruits weight and length with correlation coefficient of
Content of reducing sugar, ascorbic acid and pH values of black mulberry fruits and derived food products
Yield of juice from unprocessed fruits was from 68 to 72% and from 58 to 69% after pre-heating of fruits at 75°C. Similar results we reported by Yilmaz et al. (2012) [24], who tested the selected mulberry genotypes and obtained the juice yield of 39 –72%.
Total amount of reducing sugars was from 6.50 mg.kg–1 in wine to 60.01 mg.kg–1 in jam. Low concentration of reducing sugars in wine could be explained with the transformation of sugars into alcohol. Jam, jelly and syrup processing needs the addition of sugar to the product for preservation purposes. The differences between the technologies of producing of different kind of product could be a reason for differences in sugar content. Ascorbic acid content ranged from 0.27 mg.100 g–1 in wine to 1.10 mg.100 g–1 in fruits in honey without sterilization process. In all thermally treated products the amount of ascorbic acid was less than in unprocessed foods (Table 3). In all tested food products the pH value was acidic and varied from 3.28 in jelly to 3.69 in liqueur. Previously reported pH value for juice made from
Content of anthocyanin and free radical scavenging activity in black mulberry fruit
Mulberry is highly esteemed on the soft drink market, although the biological and pharmacological effects are still only poorly defined and the anthocyanins is highlighted as–the most important components [25]. The content of anthocyanin was determined in selected seven genotypes of black mulberry fruit - MN-1, MN-2, MN-26 with big fruit weight and genotypes MN-14, MN-31, MN-6 a MN-17 with small fruit weight (Table 1). In tested fruit samples the anthocyanins content ranged from 138.0 to 254.0 mg.dm–3 (Table 4). Genotypes with bigger fruit weight showed lower amounts of anthocyanin content (138.0 –178.0 mg.dm–3) in comparison to the genotypes with smaller fruit weight (166.0 –254.0 mg.dm–3). Differences between the genotypes were significant and the content of the anthocyanin was significantly higher in genotypes with lower fruit weight (
Detection of free radical scavenging activity showed strong effect of black mulberry fruit to eliminate the synthetic radical with values ranged from 63.00 to 72.92% (Table 4). However, the significant differences in free redical scavenging activity between the bigger and smaller fruits were not identified (
The antioxidant properties and phytotherapeutic effects of black mulberry fruits are caused by the presence of significant concentration of anthocyanins. Our study confirms that the fruit is a valuable source of natural antioxidants; however, instability during the processing and storage must be taken into account. Heating cause partial degradation and polymerization resulting in a decolourization of the product [24–28].
Anthocyanin content and free radical scavenging activity in black mulberry food products
In black mulberry-derived food products the anthocyanin content ranged from 21.4 mg.dm–3 in wine to 106.4 mg.dm–3 in fresh juice produced using kitchen juicer (Table 5). Thermal processing strongly affect the products resulting in lower concentration of anthocyanin –in juice produced by steam it decreased from 106.4 to 74.3 mg/dm3 FM, after sterilization of fruits in honey the decrease from 92.1 to 49.7 mg.dm–3 FM was observed. In the case of syrup the final content was 46.9 mg.dm–3 FM, in liqueur 37.7 mg.dm–3 FM and/or in compote 23.8 mg.dm–3 FM, and the differences in content of anthocyanon between the raw material and processed products were significant (
Mulberry fruit contain the high amount of anthocyanins and it is generally used for nutritive and therapeutic purposes. It has been reported that cyanidin 3-rutinosid and cyanidin 3-glucosid of
Free radical scavenging activity in black mulberry food products (Table 5) ranged from 50.81% in berry wine to 74.02% in sterilized fruits in honey. High level of free radical scavenging activity in fruit in honey is strongly influenced by honey, which is rich for antioxidant compounds and also compounds with antimicrobial activity [37, 38]. Generally could be concluded, there is no direct correlation between the content of anthocyanin and the antioxidant activity of tested food products. The antioxidant activities of mulberry fruit and related products could be explained with the presence of other components with they share like the phenolic acid, vitamins, and mineral compounds. Similar findings also reported Kamiloglu et al. (2013) [39], who compared antioxidant activity and content of polyphenol in fresh black mulberry fruit with related products like dried fruit, ice cream, molasses, juice, syrup, wine. Kamiloglu et al. (2013) [39] found significantly higher value of anthocyanins, flavonoids, polyphenols and antioxidant activity in fresh fruits in comparison to processed products. In future, for better understanding of pharmacokinetics of plant is necessary to intensify the studies, which are oriented on the mode of absorption and effect of mulberry antioxidants on the organisms. This will be helpful to evaluate the beneficial potential of this plant for human health.
Conclusion
Results from this study confirm that the black mulberry
Footnotes
Acknowledgments
This work was supported by the Operational Programme Research and Development of the European Regional Development Fund in the frame of the project “Support of technologies innovation for special bio-food products for human healthy nutrition” ITMS 26220220115. The authors also gratefully acknowledge to Alex Oravec and Jana Holecyova for making the photos.
