Study on the antimicrobial activity of Ethanol Extract of Propolisagainst enterotoxigenic Methicillin-Resistant Staphylococcus aureus in lab prepared Ice-cream

The objective of this study was to investigate the antimicrobial activity of ethanol extract of propolis against enterotoxigenic strain of MRSA which inoculated into lab prepared ice cream. EEP was added to ice cream in 3 concentrations (150, 300 and 600 mg/L). The prepared ice cream was divided into 2 groups, one stored at freezer temp. at (-5˚C), while the other was kept in deep freezer temp. at (-20˚C). MRSA could not be counted from the 4th, 2nd and 1st week of storage at freezer temp, while at deep freezer temp. MRSA could not be enumerated from the 3rd, 1st week and 3rd day of storage in portions contained 150, 300 and 600mg/L EEP, respectively.


Introduction
extensively used, as phytochemical ingredient, in functional foods at levels that may confer health In the last few decades staphylococcal food benefits (International Food Information Service, poisoning has been reported as the third cause of 2005).Propolis chemical composition is complex foodborne illness in the world (Zhang et al., and varies according to its botanical and phyto-1998).Staphylococcus can usually be treated geographical origin, but in general, propolis in with antibiotics, but some strains of Staphylococci nature is composed of 30% wax, 50% resin and have become resistant to antibiotics that once vegetable balsam, 10% essential and aromatic destroyed it (WebMD, 2010) (Kluytmans et al., 1995).
responsible for the low incidence of bacteria and In the context of the lack acceptability of moulds within the hive as it has antibacterial and synthetic preservatives, there is a growing antifungal properties.(Bankova et al., 2000).The interest of introducing natural additives to food.
antimicrobial effect of propolis is due to its Propolis is an interesting alternative to be considered components that are mostly of phenolic nature, in new applications of food technology as it is mainly flavonoids, as the simple phenols, phenolic The extract was transferred to small glass vials acids and polyphenols are active antimicrobial with a small amount of methanol which evaporated.agents (Cowan, 1999 andIshida et al., 2011) final count of 7×10 CFU/ml, then mixed and of propolis extract against MRSA, so this study poured in Petri dishes and left for 1 h to solidify.was performed to investigate the effect of ethanol extract of propolis on MRSA inoculated into lab Detection of MIC: MIC was determined using prepared ice cream, as Staphylococci might agar disk diffusion method (150 mg/L).contaminate ice-cream from non-hygienic serving Manufacturing of ice cream in lab: Ice cream tools and handlers.
powder was added to 2 litre of cool sterilized milk

Materials and Methods
then manufactured according to the label of instructions of the produced company.Two ml of Collection and extraction of propolis: Propolis the previously prepared MRSA cellular suspension samples were collected from different regions of 8 (7×10 CFU/ml) were added to ice cream and Egypt.The obtained crude propolis was stored at 5 o mixed thoroughly, to obtain initial count of 7×10 -18 C in a domestic freezer till its use.The frozen CFU/ml.Ice cream was divided into 4 portions propolis was grounded using a blender as and 3 concentrations (150, 300 & 600mg/L) of described by Haddadin et al., (2008).Propolis th EEP were added to 3 portions and the 4 one was samples were extracted according to the method kept as a control (EEP free).Each portion was described by Biscaia and Ferreira (2009).5 g of divided into 2 portions, one was kept at freezer propolis were placed inside a paper timber and

Results are shown in Table-1 and Table-2.
The data presented in Table-1 showed Recently, consumers think that the natural food that, The count of MRSA in control samples was preservatives are better and safer than synthetic 4 decreased gradually from (2×10 CFU/g) to ones as they are considered the reason of many 3 th (3.8×10 CFU/g) at the 4 week.The count of carcinogenic and teratogenic attributes as well as Methicillin-resistant S.aureus, in ice-cream sample residual toxicity.The investigation of natural portion contained 150mg/L ethanol extract of sources, which offer an unique pool of chemically propolis (EEP) and stored at freezing temp.at diverse substances, is a valid approach to the 3 zero time was (8.7×10 CFU/g) with (65.5%) search for new antimicrobials with chemical reduction percent then reduced gradually to reach scaffolds that differ from known antibiotics rd (70 CFU/g) at 3 week with reduction percent would have less tendency to generate microbial th (99.2 %), while through the 4 week MRSA was resistance and health problems.In the last showed no visible colonies to be enumerated but decades, propolis has gained wide acceptance by could be isolated.In the ice cream portion with people from many western and eastern countries.concentration of 300mg/L EEP MRSA count was The effect of propolis samples that were collected 3 reduced gradually from (2×10 CFU/g) at Zero by Raghukumar et al., (2010) against MRSA was 2 time to (1.2×10 CFU/g) with reduction percent of so much higher than that obtained by our propolis st (99.8%).At 1 week count of MRSA was extract (MIC= 64mg/L), while the antiMRSA 2 effect of 2 of 3 proplis samples groups collected diminished gradually from (8.6×10 CFU/g) at

o submitted to 6 h
Soxhlet extraction at a maximum (0±2 C) and the other was stored at deep freezer o o temperature of 60 C, using 150 ml of solvent (-20 C).Samples were taken to detect the initial (ethanol).Waxes from extract were removed by 3 count before addition of EEP and after hardening o consecutive steps of maintaining at -18 C overnight of ice cream portions at the first 3 days and then o and filtration at 0 C. The resulting extracts were weekly for 4 weeks for screening the antimethicillinevaporated at reduced pressure at a low temperature resistan Staphylococcus aureus (MRSA) activity o (<40 C) in a rotary evaporator to afford a of EEP.The MRSA count/g was determined using concentrated ethanolic extract of propolis (EEP).pour plate technique on BHI agar.

Estimation of MIC of EEP: Estimation
Silva, et al., 2008acterial, antifungal and antioxidant performed according to method (disk diffusion properties of propolis are combined with the fact method) prescribed bySilva, et al., 2008.that several of its constituents present in food

Preparation of MRSA cellular suspension: and
/or food additives, and generally recognized Enterotoxigenic MRSA strain was obtained from as safe (GRAS)(Burdock, 1998), make it an Food Hygiene Department, Faculty of Veterinary attractive candidate as a natural preservative in Medicine, Assiut University.The inocula was new food applications.This meets the demand for prepared from MRSA pure culture, incubated in natural antioxidants and antimicrobials, fueled o Brain Heart Infusion (BHI) agar for 24 h at 35 C. by the increasing consumer awareness for One milliliter of the culture was serially diluted to natural, minimally processed foods with obtain the count of the organism/ml.One tenth traditional preservatives absent or at very low (0.1) ml of the obtained cellular suspension concentrations(Han and Park, 1995 and Tosi et 8 (7×10 CFU/ml) was added to 100 ml of brain al., 2007).Kilic et al., (2005) and Raghukumar et o heart infusion agar, tempered at 45 C, to obtain a al., (2010) documented the antimicrobial activity 5

Table - 1: Effect of different concentrations of ethanol extract propolis (EEP) on enterotoxigenic Methicillin- o Resistant Staphylococcus aureus (MRSA) in lab prepared ice cream stored at freezing temperature
(-5 C).

Table - 2: Effect of different concentrations of ethanol extract propolis (EEP) on enterotoxigenic Methicillin- Resistant Staphylococcus aureus (MRSA) in lab prepared ice cream stored at deep freezing temperature (-20°C)
Faculty of Veterinary Medicine, EEP/L and stored at freezing temperature (-5°C), Assiut University and Dr. Moustafa Fawzy Aly, respectively.Professor of Organic Chemistry, Chemistry From the data illustrated in Table 2 it is Department, Faculty of Science, South valley evident that, the antiMARSA effect of propolis in University for their help in propolis extraction.ice cream contained 150mg/L EEP and kept at Also we are thankful to all members of Food Hygiene deep freezing temperature reduced the count of 3 Department, Faculty of Veterinary Medicine in MRSA from (3×10 CFU/g) to (80 CFU/g) during both Assiut and South Valley Universities. 2 weeks.MRSA count, in ice cream portion with 2 added 300 mg/L EEP, diminished to (1×10