Antimicrobial testings , gas chromatographic analysis and olfactory evaluation of an essential oil of hop cones ( Humulus lupulus L . ) from Bavaria and some of its main compounds

Department of Clinical Pharmacy and Diagnostics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria University of Food Technology, Department of Microbiology, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria University of Food Technology, Department of Essential Oils, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria Kurt Kitzing GmbH, Hinterm Alten Schloss 21, D-86757 Wallerstein, Germany SHIMADZU Germany, Department of GC and GC/MS, Albert-Hahn-Strasse 6-10, D-47269 Duisburg, Germany


Introduction
In continuation of our research work in the field of combined data interpretation of antimicrobial testing (agar diffusion and agar dilution methods) and gas chromatographic analysis (GC and GC-MS) as well as olfactory evaluation of aroma samples, including pure compounds, essential oils and extracts [1][2][3][4][5][6][7][8][9][10], a sample of essential oil of hop cones ( Humulus lupulus L., Cannabaceae) from Bavaria was investigated.
Despite the importance of hop and its essential oil for the aroma and bitterness of beer production, few data of biological activities such as sedative effects caused by prenylflavonoids [21], as well as antimycobacterial [22] and antibacterial [23] effects were published, but no detailed information about the influence of some main and minor compounds of the essential oil on their antimicrobial activities are available until now.
Therefore, the objectives of this research work were to analyze the composition of the essential hop oil from Bavaria by using gas chromatographic methods (GC and GC-MS), to evaluate olfactorially the sample, and to compare this data with aroma impressions of the constituents.The essential H. lupulus oil and some main and minor compounds were also tested for antimicrobial activities by means of the usual methods agar diffusion and agar dilution with various strains of microorganisms [1][2][3][4][5][6][7][8][9][10].
As result of the combined data interpretation, the above mentioned influence of the main and minor components on the antimicrobial effects of the essential hop oil can be ascertained.On basis of these data, the systematic investigation of aroma compounds and odorous samples (essential oils and extracts) will be continued in a running, international project.

Results and discussion
The essential oil of Humulus lupulus L. cones from Bavaria was olfactorially evaluated by perfumers and aroma-chemists as follows: Herbal-green, spicyaromatic, somewhat bitter and sweet with connotions of valerian.
To control the efficiency of the used microbiological testing methods, eugenol as a phenolic compound with well-known antimicrobial activity against many strains of microorganisms was tested.Eugenol shows excellent effects against all strains using both methods, agar diffusion and agar dilution (see Table 1 ).
In addition, the synthetic antibiotics Ciproxin R , Lidaprim R and tetracycline hydrochloride were used as reference substances for the antimicrobial tests.
Tetracycline hydrochloride was effective against all Gram-positive and Gramnegative bacteria, surprisingly Ciproxin R showed no antimicrobial activity against Salmonella sp. and Lidaprim R against Klebsiella pneumoniae ( Table 1 ).
The essential oil of Humulus lupulus L. cones from Bavaria showed high activity against both Gram-positive bacteria ( Staphylococcus aureus and Enterococcus faecalis), the Gram-negative bacteria Escherichia coli and Salmonella sp.) and the yeast Candida albicans, while no effects were observed against the Gram-negative-bacteria Pseudomonas aeruginosa, Proteus vulgaris and Klebsiella pneumoniae ( Table 1 ).
The monoterpenic target-compound myrcene was found to possess high antimicrobial activities only against Escherichia coli and Proteus vulgaris, but showed no effects against all other microorganisms of the test-series.Medium antimicrobial effects of the sesquiterpenic hydrocarbon -humulene against the Gram-positive bacteria Enterococcus faecalis and Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Salmonella sp. was found, whereas for the second tested sesquiterpenic hydrocarbon -caryophyllene high antimicrobial activities against the Gram-negative-bacteria Pseudomonas aeruginosa, Proteus vulgaris and Salmonella sp. as well as medium effects against the Gram-positive bacteria Enterococcus faecalis and the Gram-negative bacteria Escherichia coli could be observed.The monoterpenic hydrocarbon -pinene showed high/medium antimicrobial effects against both Gram-positive bacteria negative-bacteria Escherichia coli and Proteus vulgaris as well as a medium one against Candida albicans.With the monoterpenic alcohol geraniol the only targetcompound with medium up to high antimicrobial effects against all microorganisms was tested.Isobutyl isobutyrate was the second target-compound with medium up to high activities against all microorganisms except for the Gram-positive bacteria Staphylococcus aureus, where no effects were found (see Tab. 1. Antimicrobial activities of the essential hop oil from Bavaria, some constituents and reference compounds Using gas chromatography (GC-FID and GC-MS with 2 columns of different polarity) in combination with olfactoric evaluations, the purity of the target compounds as well as the composition of the essential hop oil was investigated.
In general, the purity of all tested components was found to be higher than 96% with a range from 96.8% for -pinene up to 99.7% for -humulene.The olfactoric evaluation with characteristic attributes for each sample proves the high purity and quality of these compounds (see

Tab. 2. GC analysis and olfactoric evaluation of target compounds
The gas chromatographic investigation of the essential oil of Humulus lupulus L.
The olfactory data for each constituent of the essential hop oil by using the description given by perfumers and aroma-chemists as well as the correlation with published odor attributes [24][25][26][27][28] is also given ( Table 3 ).
The odor-attributes of main and minor compounds of the essential H. lupulus oil are responsible for the characteristic odor-impression of this sample and additionally proof the high quality of this essential hop oil from Bavaria.
As a final remark of this combined investigation of antimicrobial activities, gaschromatographic (GC-FID and GC-MS) analysis and olfactory analysis of the essential oil of Humulus lupulus L. cones from Bavaria and some target compounds, we can state that the sample was described with a characteristic odor for essential hop oils and as main compounds myrcene, -humulene andcaryophyllene were identified.The essential hop oil showed high antimicrobial activities against Gram-positive-bacteria St. aureus and Enterococcus faecalis, Tab. 3. Composition of the essential hop oil (%-peak area of GC using an apolar fused silica OV-5-type column)

Samples
The essential hop oil is a product of Kurt Kitzing Co., Germany as follows: Fresh cones of Humulus lupulus L. (Cannabaceae) from Bavaria (product-no 800387).Cultures, Sofia, Bulgaria -were used.
The antimicrobial activity was studied by two methods: Agar diffusion disc method using Whatman No. 1 filter paper discs (6mm) and quantities of 6 l of the sample each.After cultivation of the bacteria and the yeast at 37°C for 24 h the diameter of the inhibition zone (IZ) was measured [1][2][3][4][5][6][7][8][9][10] as well as agar serial tube dilution method which results as minimum inhibitory concentration (MIC) in accordance to [1][2][3][4][5][6][7][8][9][10] as follows: The essential oil and reference compounds were added to brine, containing 1.0% (v/v) Tween 80 at the appropriate volumes to produce final concentrations of the samples in the range of 1-1000 ppm; the Petri dishes were inoculated by pipetting 0.1cm 3 of the desired culture and 6 µL of the samples as well as the reference compounds (the tablettes of Ciproxin R were added as solution in saline at a quantity of 300 µg and 30 µg) on paper discs (6 mm) and then incubated at 37°C for 24h.

Olfactoric evaluation
All investigated samples were olfactorically evaluated by professional perfumers and/or olfactively trained chemists and the aroma described as mentioned in Table 1 and 2 and correlated with published odor impressions [24][25][26][27][28].
A comparison of these data show that the antimicrobial effects of the essential hop oil against Staphylococcus aureus can be correlated only to the constituents geraniol and -pinene and the effectivity against the yeast Candida albicans to geraniol, isobutyl isobutyrate and -pinene.The antimicrobial activities of the essential H. lupulus oil against Enterococcus faecalis, Escherichia coli and Salmonella sp.results of medium up to high effects of some target-compounds.Staphylococcus aureus, Enterococcus faecalis and a high activity against the Gram-* no inhibition observed
*relative %-peak-area using GC-FID with an a apolar OV-5-column or b polar carbowax-column

spicy, somewhat oily -Humulene 1455 31.2 soft-woody, reminding to fresh earth
-negative-bacteria Escherichia coli and Salmonella.sp. as well as the yeast Candida albicans.These effects do not result from antimicrobial activities of a single constituent, but from the combination of main and minor compounds.