Volatile composition of Anatolian propolis by headspace-solid-phase microextraction ( HS-SPME ) , antimicrobial activity against food contaminants and antioxidant activity

Propolis is an important natural resource utilized by humans since ancient times. It is a resinous mixture containing polyphenols, mainly flavonoids and phenolic acids as well as volatile compounds. Propolis has been used for its antibacterial, antifungal, antiviral, antineoplastic, antioxidant, immunomodulating and antiinflammatory properties. Ethanol extracts of three different propolis samples collected from North East Anatolia were chemically and biologically evaluated. Volatile compounds of the samples were analysed by headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). The main volatile constituents were identified as phenyl ethyl alcohol (7.7%), benzyl alcohol (7.4%), decanal (6.7%), ethyl benzoate (6.5%), nonanal (5%) and cedrol (4.1%) for the Yesilyurt propolis; cedrol (15.6%) for Sarıcicek propolis and αbisabolol (14.3%), cedrol (7%), δ-cadinene (5.6%) and α-eudesmol (3.6%) for the propolis from Erzincan, respectively. The best antioxidant activity with IC50 value of 4.95 μg/ml was defined using the spectroscopic (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay. The highest antimicrobial activity was determined by agar dilution method against the pathogen Bacillus cereus (0.06 to 0.12 mg/ml). Propolis could be a salubrious additive for the production and protection of functional foods having a microbial deterioration potential.


INTRODUCTION
Foods, whether fresh, prepared or even preserved, are rarely sterile and may be contaminated with spoilage microorganisms or sometimes with pathogens.Among other factors, microorganisms are important spoilage agents in foods (Gram et al., 2002).Microorganisms that are found on the foods may also cause food poisoning *Corresponding author.E-mail: esin.kocabas@ege.edu.tr,esinkocabas@gmail.com.Tel: +90.232.3884955. Fax: +90.232.3884955(also called food intoxication) or food infections.While food poisoning results from ingestion of foods containing preformed microbial toxins, food infections originate from ingestion of foods containing viable pathogens which cause infection and disease in humans.Staphylococcus aureus is one of the major causes of intoxication whereas Salmonella enterica, pathogenic Escherichia coli strains and Bacillus cereus are the other major causes of food infections.Various physical and chemical techniques can be used for protection of foods depending upon the type and amount of the foods (Tosi et al., 2007).
Propolis (from the Greek "pro", in defence of, and "polis", city) is a complex natural substance produced by honeybees (Apis mellifera L.) by using various plant sources and utilised by bees to seal holes in their hives and protect the hive against invaders (Sforcin and Bankova, 2011).Analysis of several samples from different geographic regions showed that both chemistry and biological activity of propolis were highly variable (Bankova, 2005b).Propolis possesses a wide range of biological activities, including anti-cancer, antimicrobial, antioxidant and antiinflammatory among others (Luo et al., 2011).
One of the applications of propolis in the food industry is the production of functional foods that are resistant to spoilage and promote health.Functional foods are an emerging field in food science targeted to provide health benefits through the prevention, management and treatment of diseases and also to extend the shelf life of food by reducing the existence of spoilage microorganisms (Milner, 2000).
The chemical composition of propolis has been extensively studied by using different methods, mainly such as gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography mass spectrometry (HPLC-MS) (Alencar et al., 2007;Luo et al., 2011;Popova et al., 2005;Righi et al., 2011).
Headspace-solid-phase microextraction (HS-SPME) offers an efficient alternative sampling and extraction technique for the analysis of volatile organic compounds.The adsorption is performed by immersing the pre-coated fibre in a liquid sample or exposing it to the headspace above a liquid or solid sample.In the last two decades, HS-SPME has found applications in environmental, biological, food and pharmaceutical analyses (Cserháti, 2010;Tasdemir et al., 2003).To the best of our knowledge, there is only one previous study on the volatiles of propolis using HS-SPME (Yildirim et al., 2004).
The aim of this study was to evaluate the antioxidant and antimicrobial activities of three Anatolian propolis extracts against important food spoilage and/or pathogenic microorganisms and to characterise their volatile constituents by the combined techniques of HS-SPME and GC-MS.

Preparation of ethanol extracts
Eight (8) grams of each propolis sample were coarsely grained and mixed with 40 ml ethanol (96%) in a 100 ml size Erlenmeyer's flask for extraction at room temperature at 150 rpm for 18 h using an orbital shaker.The extract was filtered twice using Whatman No.1 filter paper to filter the solid material.The solvent was removed using a vacuum evaporator (< 40°C).

HS-SPME of the volatile fraction
The manual SPME device (Supelco, Bellafonte, PA, USA) with a fibre pre-coated with a 65 µm thick layer of polydimethylsiloxane/divinylbenzene (PDMS/DVB-blue) was used for extraction of the volatiles.The vial containing the sample was sealed with parafilm.The fibre was pushed through the film layer for exposure to the headspace of the extract for 15 min at 50°C.The fibre was then inserted immediately into the injection port of the GC-MS for the desorption of the adsorbed volatile compounds for analysis.

GC-MS analysis
The GC-MS analysis was carried out using an Agilent 5975 GC-MSD system (SEM Ltd.Istanbul, Turkey).An Innowax FSC column (60 m × 0.25 mm, 0.25 µm film thickness) was used with helium as the carrier gas (0.8 ml/min).GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min, and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min.
Split ratio was adjusted at 40:1.The injector temperature was set at 250°C.Mass spectra were recorded at 70 eV.Mass range was from m/z 35 to 450.

Identification of components
Identification of the volatile components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention indices (RRI) to a series of n-alkanes (C9-20).Computer matching against commercial libraries (Adams Library, Wiley GC/MS Library, MassFinder 3 Library) and in-house "Baser Library of Essential Oil Constituents" built up of genuine compounds and components of known oils, as well as MS literature data was used for the identification (Tasdemir et al., 2003;Demirci et al., 2007;Polatoglu et al., 2012).

DPPH radical scavenging activity
Free radical scavenging capacity of ethanol extracts was determined using DPPH (Sigma-Aldrich, Steinheim, Germany) (Choi et al., 2006).Reaction mixtures containing 0.5 ml of 0.3 mM DPPH in ethanol and different dilutions of propolis extracts (15.6 to 500 µg/ml) in 1 ml ethanol were shaken and left in a dark place at room temperature for 20 min.The absorbance was measured at 514 nm with disposable plastic half-micro cuvettes using a Ultraviolet and visible absorption spectroscopy (UV-VIS) spectrophotometer (Jenway 6400 Bibby Scientific, UK).Results were expressed as a percentage of radical scavenging activity using the following equation:

DPPH radical scavenging activity (%) = 100 [(A sample -A blank ) × 100] / A control ]
Ethanol was used to adjust the spectrophotometer, ethanol (0.5 ml) plus propolis extract solution (1 ml) was used as the blank and DPPH solution (0.5 ml) plus ethanol (1 ml) was used as the negative control.Experiments were carried out in duplicate and results were reported as IC50 (required concentration to scavenge 50% of DPPH radicals).Ascorbic acid and quercetin were used as reference standard compounds (0.4 to 250 µg/ml).
The same test was carried out using gentamicin sulphate and nystatin (Sigma-Aldrich, Saint Louis, USA) as a positive control for bacteria and yeasts, respectively.Minimum bactericidal concentration (MBC) was determined by re-inoculating the cultures onto propolis free agar plates.MIC and MBC were defined as the lowest concentration that allowed no visible growth on the agar plates.Two replicates were made for each concentration of all propolis extracts.

Statistical analysis
The data obtained from the radical scavenging assay of the different propolis samples were compared by one-way analysis of variance (ANOVA) using statistical package for social sciences (SPSS) software (New York, USA) 10.0.The level of significance for statistical tests was p ≤ 0.05.

Volatile constituents of the ethanolic extracts of propolis samples
In the present study, propolis S exhibited the best extractive yield of 53.7% followed by propolis Y (51.2%) and propolis E (44.6%).All samples were stored at +4°C in amber bottles until further use.HS-SPME coupled with GC-MS was used for the characterisation of the volatile compounds of propolis extracts.The profile of volatile compounds is shown comparatively in Table 1.A total of 118 volatile components were identified from the ethanol extracts of three propolis samples originated from North Eastern Anatolia and Black Sea Region of Turkey.In particular, the HS-SPME of the ethanol extract of propolis Y contained 54 volatile compounds, accounting for 73.7% of the whole of the volatiles.The principal constituents of this extract were identified as phenyl ethyl alcohol (7.7%), benzyl alcohol (7.4%), decanal (6.7%), ethyl benzoate (6.5%), nonanal (5%) and cedrol (4.1%), having a pleasant odour.
Seventy four individual volatile compounds were also identified for propolis S. The sole major volatile constituent of this extract was cedrol (15.6%), having a woody and spicy characteristic smell.The third propolis sample, namely propolis E, contained 72 volatile compounds, accounting for 89.2% of the whole of the volatiles.α-Bisabolol (14.3%), cedrol (7%), δ-cadinene (5.6%) and α-eudesmol (3.6%) comprised the major volatile constituents of this extract, resulting in a characteristic odour.According to the chemical class, the volatile constituents of propolis S and E were mainly composed of oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes accounting for 68.0 and 81.8% of the whole of the volatiles, respectively.Propolis Y included 24.8% of these volatile compounds (Figure 1).

Radical scavenging activity
The free radical scavenging activity of propolis extracts was photometrically evaluated based on the reduction of DPPH in the presence of different extract concentrations.
The IC 50 values calculated by linear regression of plots are presented in Figure 2. The highest radical scavenging activity was obtained with propolis Y, with IC 50 values of 4.95 µg/ml.This was followed by propolis S with IC 50 values of 20.78 µg/ml and propolis E with IC 50 values of 37.09 µg/ml.
The IC 50 values of ascorbic acid and quercetin were determined as 4 and 2.9 µg/ml, respectively.Antioxidant activity of propolis Y was slightly lower than that of ascorbic acid.

Antimicrobial activity
As shown in Table 2, the highest MIC and MBC values were against endospore forming species, as B. cereus (0.06 to 0.12 mg/ml).An MIC value of 0.25 mg/ml, indicative of a considerable antimicrobial activity, was obtained against Methicillin-resistant Staphylococcus aureus (MRSA) for propolis Y, S and E.Among the samples, propolis Y exhibited the most efficient antimicrobial profile towards the tested microorganisms, followed by propolis S and E. Generally, ethanol extracts of propolis were determined as more active against Gram positive bacteria.Propolis Y has shown an inhibitory and bactericide activity against all yeast strains with concentration range of 0.25 to 1 mg/ml.Propolis S, a similar inhibitory effect against yeasts whereas propolis E has only shown inhibitory effect against C. albicans ATCC 10231.

DISCUSSION
In recent years, the diverse bioactivity and efficiency of propolis has encouraged research and further analysis of propolis to determine potential new applications.The chemical composition of propolis samples depends on the local plant flora, and geographic and climatic characteristics of the area where the sample was collected.Different propolis samples could be completely different in their chemistry and biological activity (Bankova, 2005b).In the current study, this chemical variation can also account for the variety of volatile compounds in the three evaluated samples.Propolis samples have different plant origin due to different geographical position.Propolis Y was collected from the Eastern Black Sea Region and possess highly diverse flora consisting spruce, beech, fir, mountain alder, chestnut, hornbeam, oak, silver birch, ash, trembling poplar and willow.Main constituents of propolis Y were detected as phenyl ethyl alcohol (7.7%), benzyl alcohol (7.4%), decanal (6.7%), ethyl benzoate (6.5%), nonanal (5%) and cedrol (4.1%).Chemical constituent, antimicrobial and antioxidant activity of propolis Y was significantly different from propolis S and E.
Plant origin of propolis S is composed mainly of pine coniferous tree such as spruce and yellow pine.The major volatile constituent of its extract was cedrol.In accordance with this, cedrol is a sesquiterpene alcohol found generally in cedar wood and other conifer essential oils.Cedrol was also reported by Melliou et al. (2007) as a major component (4.3 to 6.3%) of Greek propolis samples.Cedrol (6.3%), δ-cadinene (8.4%) and αeudesmol (12.1%) were also reported previously as major components of Greek propolis (Preveza, Arta and Agrinio, respectively) (Melliou et al., 2007).
Different propolis samples have been used and analysed to determine their constituents.The most often reported techniques for chemical analyses of propolis samples are GC-MS and HPLC (Bankova, 2005b;Sforcin and Bankova, 2011).A literature survey showed that the GC and GC-MS methods were generally used for the analysis of non-volatile compounds by derivatisation of propolis samples from various origins (Alcenar et al., 2007;Bankova et al., 2002;Popova et al., 2005;Sorkun et al., 2001;Uzel et al., 2005).There are inconsistencies and discrepancies in the reported analytical results, which are obtained after derivatisation, where many reported components are not possible to trace.In this present study, we could not trace any of the previous reported derivatised products using the same techniques.
HS-SPME-GCMS has been utilised efficiently in the analysis of volatile compounds from various matrixes in the past (Cserháti, 2010;Michel and Buszewski, 2008).The technique is fast, handy and efficient to perform a realistic analysis of the volatiles of the test samples in a non-destructive manner.Consequently, in the current study, HS-SPME-GC-MS was implemented successfully for the analysis of volatiles of Anatolian propolis samples.
The results indicate that propolis samples exhibited strong free radical scavenging activity.In a previous study, Sawaya et al. (2009) evaluated the antioxidant activity of three different Scaptotrigona propolis samples.Their findings showed that the antioxidant activities of the samples ranged between 43 and 1000 µg/ml.The authors mentioned that Scaptotrigona propolis present a much lower antioxidant activity than samples of Apis mellifera propolis.The antioxidant activity of propolis from Argentina was reported with IC 50 values of 25 to 37.5 µg/ml (Isla et al., 2009).These results are in accordance with our experimental data.The propolis from Korea also had strong DPPH free radical-scavenging activity (Choi et al., 2006).Propolis contains a wide variety of phenolic compounds, mainly phenolic acids and flavonoids.Indeed, flavonoids and various phenolic compounds have already been studied as antioxidants and demonstrated to be very active (Isla et al., 2009;Righi et al., 2011).A correlation between the antioxidant activity and total polyphenol and flavonoid content of propolis was also reported (Choi et al., 2006).
Microorganisms are the major deteriorating agents of food and also causative agents of severe food-borne illness (Tosi et al., 2007).In the field of functional food, some additives such as flavonoids can be used against food-borne pathogens and also increase the potential positive effect of the food on health (Luo et al., 2011).Therefore, the inhibitory potential of propolis against a set of pathogenic microorganisms was evaluated.The representative major food spoilage and/or pathogen microorganism of three gram positive, three gram negative bacteria and four yeast species were used (Table 2).Among the test organisms, two of them were selected from antibiotic resistant species including MRSA and VREF.
It is well-known that B. cereus is adapted for growth in the intestinal tract of insects and mammals.From these habitats, it is easily spread to foods, where it may cause food-associated illness by the production of diarrhoeal or emetic enterotoxins (Stenfors, et al., 2008).In addition, endospores of the Bacillus sp. have extreme resistance to heat and other preservation treatments (Caspers et al., 2011).The most important bacterial pathogen of the honey bee is endospore forming Paenibacillus larvae, which causes severe brood diseases.Therefore, bees need to be protected primarily against the invasion of this pathogen.Probably due to the strategy developed by bees against endospores of P. larvae, propolis can be effective in a similar way on Bacillus endospores.In a recent study, Mihai et al. (2012) reported the significant inhibition of P. larvae treated with ethanol extracts from different propolis in vitro using solid agar diffusion technique.In the current study, we observed significant antimicrobial effect of the ethanol extract of propolis against endospore forming B. cereus.The lower efficiency of the propolis extracts against gram negative bacteria may be dependent on the membrane structure of gram negative bacteria (Probst et al., 2011).Reports showed that propolis has previously shown no or limited activity against gram negative bacteria (Popova et al., 2005;Righi et al., 2011).
The MIC values of the ethanol extract of Brazilian propolis were reported as 1.54 ± 0.62 mg/ml for S. aureus and 19.24 ± 2.2 for E. coli (Probst et al., 2011).A previous study reported that the ethanol extract of red propolis had antibacterial activity against S. aureus ATCC 25923 with MIC ranging from 100 to 200 µg/ml (Alencar et al., 2007).This result is close to our findings for S. aureus (MIC 0.25 mg/ml).Determination of antimicrobial activity by disc diffusion in mm or using extracts solution (%) versus µg/ml or mg/ml values complicated the comparability of results (Choi et al., 2006;Mihai et al., 2012;Popova et al., 2005).Moderate antimicrobial activity of the propolis, especially against gram positive bacteria can be seen in several reports.
The antimicrobial efficiency of propolis is not a surprising phenomenon considering that this natural compound is produced by bees to defend against bacterial and fungal infections (Bankova, 2005a).The antimicrobial efficiency of propolis samples was attributed to their high content of flavones and flavanones (Choi et al., 2006;Righi et al., 2011).Propolis, a defence and protection shield produced by bees against their pathogen P. larvae and other invaders, can be a powerful antimicrobial candidate against B. cereus strains, which are highly problematic foodborne pathogens and/or spoilage microorganisms.Considering additionally the significant antioxidant activity of propolis, this natural product could be a useful additive for the production of functional foods, especially where foods have the potential for B. cereus contamination.

Table 1 .
Volatile components of three propolis samples as determined by HS-SPME and GC-MS.

Table 2 .
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the tested propolis extracts.