DIFFERENT ANTIMICROBIAL EFFECTS OF RASPBERRY DEPENDING ON THE METHOD OF ACTIVE COMPONENTS ISOLATION

Raspberry fruit exhibits numerous beneficial effects for veterinary and human medicine. One of those effects is antimicrobial activity, because of its diverse chemical composition. Adequate processing of plant material is the most important part of examining their activities. Processing conditions affect the separation of chemical substances in the test sample i.e. different methods can lead to separation of different active principles. Raspberry juice was produced by filtration. Raspberry pomace, the residue from the pressing of raspberries for juice production was extracted with ethanol and ether. The aim of this study was to determine the most suitable extraction procedure in order to obtain extract with the highest antimicrobial potential. The results of well diffusion method indicate that raspberry juice and ethanol extract of raspberry pomace have shown higher antimicrobial potential in comparison to ether extract.

In order to examine antimicrobial activity of all substances present in the raspberry fruit, three different extraction procedures were performed: simple filtration, extraction with polar solvent and extraction with nonpolar solvent.The aim of this study was to determine which of these methods of extraction is the most suitable for investtigating antimicrobial activity of raspberries in vitro by well diffusion method.

Test material
Raspberry cultivar Meeker was obtained from the "Elixir" factory (Šabac, Serbia).Freshly harvested fruits were processed the same day.Raspberry juice was produced by filtration through gauze and Buchner funnel into a glass bottle.After squeezing fruits, 200 ml of a mixture containing 80% of ethanol, 19.95% water and 0.05% acetic acid was poured over 200 g of residue (pomace).The mixture was treated by shaker (KS501, IKA) at 250 rpm at 25 °C for 30 minutes, and then filtered using filter paper and funnel.The procedure was repeated by adding 100 ml of solvent mixture and treated for 15 minutes.Obtained blends were evaporated to dryness on a rotary vapor (RV10, digital V, IKA). 1 g of the resulting ethanol extract was dissolved in 15 ml of sterile distilled water.After ethanol extraction, pomace was poured with 100 ml of ether and treated by shaker at 250 rpm at 25 °C for 15 minutes, and then filtered using funnel and filter paper.

Tested microorganisms
Sixteen bacterial, fungus and alga strains were used to assess the antimicrobial properties of the test samples (juice of fresh fruits, ethanol and ether extracts of raspberry pomace), including eight Gram positive bacteria (

Microbial susceptibility testing
The microbial susceptibility assay was performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006) using agar well diffusion method.The density of the suspensions of tested microorganisms was adjusted to turbidity equal to the 0.5 MacFarland standard.Wells (5 mm in diameter, volume 80 µl) made in agar were filled with a test samples, negative and positive controls.The agar plates were incubated at 37 °C for 24 hours.Antimicrobial activity of tested samples was determinated by the measurement of the zones of microbial growth inhibition.All tests were performed in triplicates.Distilled water and ether were used as negative controls, whereas streptomycin and nystatin (Galenika, Zemun, Serbia) were used as positive controls.Streptomycin (2000 µg/ml) and nystatin (6-25 µg/ml) were dissolved in physiological saline before testing.Statistical analysis was performed in Microsoft Office Excel 2007 program (Microsoft, One Microsoft Way, Redmond, VA, USA).

RESULTS AND DISCUSSION
Results are shown in Figures 1-4.For other investigated isolates we have not found comparable data in the available literature.
Similar   Difference between juice and ethanol extract's activity against P. wichermanii (Fig. 4) was not significant (p>0.05).There was no comparable data in the available literature.

CONCLUSION
The results of this study indicate the strongest antibacterial effect of raspberry juice followed by ethanol extract, while ether extract of raspberry pomace did not shown antimicrobial activity against any of the tested microorganisms.High volatility of ether make this solvent less suitable for this application and further investigation should be performed with some less volatile solvents, such as methylene chloride or tetrahydrofuran.Although the results of this study are generally similar to previous studies, differences in antimicrobial potential of the tested extracts could possibly be explained by: different chemical composition of raspberry, geographic origin, time of harvest, method of storage and extraction, as well as from differences in the genetic characteristics of the tested strains.

АCKNOWLEDGEMENTS
This work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Project number 46012 and 41012.

Figure 3 .
Figure 3. Mean values of inhibition zones' diameters (mm) of the test substances against the fungus (Ca-Candida albicans)

Figure 4 .
Figure 4. Mean values of inhibition zones' diameters (mm) of the test substances against the alga (P-P.wichermanii)