EurBee board Dorothea Brückner, Germany Norberto Milani, Italy Robert Paxton, Great Britain Dalibor Titěra, Czech Republic Bernard Vaissiere, France Program consultant

Bee products

Swiss Bee Research Centre, E-mail: stefan.bogdanov©alp.admin.ch

The main issues in HONEY analysis at present regard general quality and authenticity. Control of honey quality is carried out according to the standards of the Codex Alimentarius and the EU using the harmonised methods of the IHC. Residues is another important issue of honey quality. There are different contamination risks, outlined recently the main problem at present being antibiotic residues (Bogdanov, Apidologie, 38, 2006). Authenticity includes 3 aspects: production, geographical and botanical origin. Honey adulteration is tested most efficiently by stable isotope analysis. The geographical origin is tested mostly by pollen analysis. The authenticity of botanical origin is examined by two methods: The classical approach by combined assessment of sensory, microscopic and chemical results. New methods like analysis of volatiles and spectroscopy are also very promising for the future. Control of BEESWAX is generally carried out according to the Pharmacopoe, but for wax adulteration gas-chromatographic methods should be used. There are no international standards for the regulation of PROPOLIS, POLLEN and ROYAL JELLY control. Drafts for propolis, pollen and royal jelly standards have been proposed by the IHC. The propolis draft includes proposals for the two main types of propolis present on the market, originating from poplar and Baccharis. They include general criteria like humidity, wax content, mechanical impurities, and also the main biologically active compounds, the polyphenols. The pollen draft for includes humidity, lipids, proteins, as well as biologically active compounds like vitamins and polyphenols. The royal jelly draft includes humidity, protein, carbohydrates, lipids (mainly 10-hydroxydecenoic acid), minerals and acidity. As these bee products are often used for health enhancement purposes, their contaminant level should correspond to the Pharmacopoe requirements.


Honey: Authenticity of botanical and geographical origin

Dr. Werner von der Ohe

LAVES Institut für Bienenkunde Celle, Herzogin-Eleonroe-Allee 5, 29221 Celle, Germany, E-mail: werner.von-der-ohe©laves.niedersachsen.de

Within the European Community price structuring of honey and other bee products depends mainly on botanical and geographical origin of the product. Honey from overseas is offered and traded at a lower price than most honeys from countries of the EC. Especially in Germany quite good prices for unifloral honeys from special regions can be fetched. Due to cover their expenses beekeepers within the EC have to rely on higher prices for their bee products. But also beekeepers form overseas selling good bee products without residues should participate of better prices which can be achieved for those products in the EC. But to guarantee the indicated origin to the consumer quality insurance and quality control it is of great importance.

In this field pollen analysis of honey, melissopalynology, is of great importance. Honey always includes numerous pollen grains (mainly from the plant species foraged by bees) and honeydew elements (like wax tubes, algae and fungal spores) that altogether provide a good ‘fingerprint’ of the environment where the honey comes from. Due to the fact that pollen of different plant species can be distinguished and flora of different regions varies pollen analysis can be useful to determine and control the geographical and botanical origin of honeys.

In case of looking for botanical origin it is also necessary to carry out sensory and physico-chemical analyses beside pollen analysis. The main criteria are sensory, consistency, colour, sugar spectra and electrical conductivity. In some cases also the amount of proteins and special aromatic components can be helpful. At present some new methods like infrared-spectroscopy are evolved and checked for meaningfulness. Bee colonies never will produce honey from only one source. So beside the main honey flow you always will have some other parts of nectar and honeydew sources in honey. The directive for honey (2001/110/EC) takes this into account. Unifloral honeys should come wholly or mainly from the indicated source. In Germany mainly was defined in the commentary of food law at least 60 %. The EC commission has pointed out that mainly is close to wholly. Other questions which can be settled in this field e.g. can blends of honeys be indicated as unifloral honeys.

The geographical origin can be distinguished by pollen analysis. But there is a problem with filtered honeys. After filtering there are no pollen grains left. At present isotope analysis are evolved. Current analysis on isotopes proportion (d13C/12C, d18O/16O, d2H/1H) shows encouraging results.

Länderinstitut für Bienenkunde Hohen Neuendorf e.V., E-mail: lichtenberg.kraag©rz.hu-berlin.de

Honey is a product with a wide range of natural compounds depending on the floral source. Pollen analysis, sensorial testing and physiochemical characterisation of a honey sample are the parts of the authentification. In order to increase sample size and reduce costs of analysis, there have been many efforts to find alternative methods. In general, the infrared spectroscopy is a useful tool in food analysis. The FTIR instrument used for our study is constructed for routine measurements and has been established for analysing most of the physiochemical parameters of honey: sugar compounds, pH, electrical conductivity and free acidity can be measured with a high reliability, for HMF and proline it is used as a screening method.

In addition, FTIR can be used for determination of the botanical origin of a honey sample by comparison of the complete mid-infrared spectra. After calibration the floral source and the physiochemical parameters can be analysed in parallel.

Honey samples from local beekeepers were collected and characterized by standard methods. The most common honey types were used for calibration.

We recorded the complete mid infrared spectrum from each honey sample. PCA-calibration models were used for comparison of the spectra. Validation was performed with samples of various botanical origins.

Honey of rape, false acacia, heather, clover, lime tree, honeydew, sunflower and cornflower was calibrated and improved during routine analysis. Most of the honey samples from rape, false acacia, heather and honeydew can be classified correctly by the FTIR in consideration of the physiochemical and sensorial properties.


Determination of botanical and geographical honey origin by electronic nose

F. Čačić¹, S. Mannino², S. Benedetti², Lj. Primorac¹, D. Kenjerić¹, A. Perl<sup>1

1</sup> Faculty of Food Technology, University of J. J. Strossmayer, Franje Kuhača 18, 3100 Osijek, Croatia.

² Department of Food Science and Technologies, Chemistry Unit, University in Milano, Via Celoria 2, 20133 Milano, Italy.

Determination of honey botanical origin is essential factor that is required prior entering any market, and it is factor that dictates price. Problems associated with botanical origin determination are rooted in fact that required analyses (pollen analysis, physicochemical parameters determination and sensory analysis) are arduous and time consuming.

This work examines performance of electronic nose based on 12 MOS and 10 MOSFET sensors in classification of different honey samples collected in Croatia with respect to their botanical and geographical origin. Data analysis is performed with StatSoft Statistica 7 software package using modules for PCA (NIPALS algorithm) and ANN. Classification results clearly demonstrates ability of electronic nose to distinguish different types of honey, and within same honey type differences that can be correlated with geographical origin.

Quality evaluation and application of response surface methodology to optimise the pH, temperature and time to establish the quality attributes of hydroxymethylfurfural and diastatic activity in Eucalyptus honey

Vikas Nanda¹ and A.K.Bakhshi<sup>2

1</sup> Department of Food Technology, SLIET, Longowal. (Punjab). India

² Department of Food Science and Technology

Punjab Agricultural University, Ludhiana (Punjab). India

In this study samples of Eucalyptus honey (Eucalyptus lanceolatus) were botanically characterized by pollen analysis. Analysis of samples for moisture, pH, hydroxymethylfurfural, diastase activity, optical rotation, mineral content and sugar composition showed that the honey samples met all the national and international standards. Response surface methodology was used to analyse the effect of temperature, time and pH on the quality responses (hydroxymethylfurfural concentration and diastatic activity) of Eucalyptus honey. A rotatable central-composite design was used to develop models for the responses. Hydroxymethylfurfural concentration was increased with increase in temperature and pH while keeping the other variables constant. Diastatic activity was decreased as the pH moved away from the optimum value of 5.2 to 5.6. Three-dimensional response surfaces were superimposed, and the regions meeting the diastatic activity calculated as diastase number (9 to 23.09°G) and Hydroxymethylfurfural concentration (3 to 10.21 mg/kg) was identified at 48±1°C for 9.5±1 minutes at 5.1±0.15 pH. These predicted values for optimum process conditions were in good agreement with experimental data.

Research Institute of Pomology and Floriculture, Apiculture Division., 24-100 Pulawy, Kazimierska 2, Poland

* National Veterinary Research Institute, 24-100 Pulawy, Al Partyzantow 57, Poland
E-mail: teresa.szczesna©man.pulawy.pl

Honey as foodstuff of animal origin is subject to constant monitoring for the presence of banned substances or for the excessive contents of substances which can be used in animals and which are eventually allowed to be present in specified amounts. In order for a given honey to be marketed the results of the control tests must agree with the provisions of the EC regulation on the establishment of maximum residue limits (MRL) of veterinary medicinal products in foodstuffs of animal origin. In the above regulation there are no residue limits for antibacterial substances (antibiotics and sulfonamides) in honey. Hence the finding of any amount of above-mentioned substances may provide the basis to disqualify this product even though the amounts of the detected contaminants are varied and the results are directly affected by the analytical methods used. Beekeepers in the Union countries have not ceased to administer drugs to control American and European foulbrood, in spite of the prohibition of their use. Research conducted in Poland also indicates that domestic honey is contaminated mainly with sulfonamide residues. It bears out the most frequent use of the sulfonamide drug – Polisulfamid (sulfathiazole, sulfacetamide, sulfamethazine) – in Polish apiaries.

The aim of our the study was to optimize the procedure for sulfonamide residues analyses in honey and their control in commercial honey. Separation, identification and quantity analysis of sulfonamides in honey were carried out using SHIMADZU HPLC instrument equipped with fluorescence detector after synthesis of sulfonamide derivatives with fluorescamine. Chromatographic conditions were performed using analytical standards of sulfathiazol, sulfacetamide, sulfamethazine and p-aminobenzoic acid (PABA), naturally present in some honeys. With these conditions p-aminobenzoic acid (PABA) was well separate from sulfamethazine. In a practical analysis limit of detection was set at 5 µg/kg for each of three analysed sulfonamides. Received results show that sulfonamide residues in commercial honey ranged from slight up to above 1000 μg/kg. To take into consideration practical detection limit (5μg/kg) sulfatiazole and sulfamethazine were detected in 50% of commercial honey samples, sulfacetamide in 16%. Total sulfonamides level in 30% of honey samples was above 50 μg/kg – quantification limit establish by Polish National Monitoring Program 2005.
Chromatographic determination of Artepillin C in Brazilian propolis

Ligia Bicudo de Almeida Muradian

University of Sao Paulo, Brazil, E-mail: ligiabi©usp.br

Propolis is a resin of varied color and consistency collected by honeybee, Apis Mellifera, from several parts of the plant such as sprouts, flower buds and resinous exsudatos, being transported to the hive with the intent of defending it. Studies performed with the Brazilian propolis showed the presence of several phenolic compounds, as the most recent brings the identification of prenyl derivative of coumaric acid. Among them, the most studied one is 3,5-diprenyl-4-hydroxycinnamic acid (Artepillin-C), isolated from the propolis produced in areas which flora is rich in Baccharis species. Its quantification has become an important factor as indicator of Brazilian propolis quality. This phenolic acid has biological properties such as antitumoral, antibacterial and antioxidant activity showing that Artepillin C can be one of the most important active compounds from Brazilian propolis. It is used as a chemical marker for the quality control and exportation of green propolis. The purpose of this work is to validate the method and evaluate the content of Artepillin C from 33 samples collected in different Brazilian regions. The method used to quantify Artepillin C is an HPLC method using RP-column (C-18) at 25oC, mobile phase with acetonitrile / phosphoric acid (0,1%) in a proportion of 50: 50 (v/v), UV-VIS detection at 280 nm and retention time at 15 min. The validation parameters studied were: linearity, accuracy, precision, quantification limit and detection limit. The results obtained for the validation parameters were: detection limit = 0.0036 ug/mL, quantification limit = 0.012 ug/mL, accuracy = 0.0064 (SD) and 0.078 (RSD), recovery of 98 to 102%. Artepillin C content varied from 0 to 11% depending on the geographical origin. The southeast region of Brazil presented the highest level of Artepillin C (5.0 – 11.0%) while the northeast region did not show any Artepillin-C.

ANTIBACTERIAL ACTIVITY OF PROPOLIS SAMPLES FROM DIFFERENT GEOGRAPHICAL REGIONS OF TURKEY AGAINST TWO FOOD-BORNE PATHOGENS, SALMONELLA ENTERITIDIS AND LISTERIA MONOCYTOGENES

Ayhan Temiz¹, Ayla Şener¹, Aslı Özkök Tüylü², Kadriye Sorkun²* , Bekir Salih<sup>3

1</sup> Department of Food Engineering,Faculty of Engneering,Hacettepe University,06532 Beytepe-Ankara,Turkey

² Department of Biology,Faculty of Science,Hacettepe University,06532 Beytepe-Ankara,Turkey

³ Department of Chemistry,Faculty of Science,Hacettepe University,06532 Beytepe-Ankara,Turkey

Corresponding author* kadriye©hacettepe.edu.tr

Antibacterial activities of 25 propolis samples collected from different regions of Turkey were investigated against two food-borne pathogens, Salmonella enteritidis (ATCC 13076, SE) and Listeria monocytogenes (ATCC 1462, LM). Ethanol extracts of the propolis samples (EEPs) were prepared and chemical compositions EEPs were determined by GC-MS. Antibacterial activities of EEPs were tested at concentrations of 10% and 1%, and evaluated according to the reductions in the viable cell number of the bacteria by using pour-plate method. All EEPs were bactericidal for both bacteria at concentration of 10%. Totally 19 EEPs had inhibitory effect on LM at 1%. Eight EEPs at this concentration completely inhibited the growth of LM whereas 11 EEPs caused 0.60-5.93 log reductions in the viable cell numbers. Fifteen EEPs had no effect against SE at 1%, other 10 EEPs had only limited effect with 0.04-0.71 log reductions in the viable cell numbers. Seven main organic compound groups were determined in EEPs; flavonoids, aromatic acid esters, aromatic alcohols, aromatic acids, aliphatic carboxylic acids, terpens, and aliphatic carboxylic acid esters. Flavonoids were the only shared component found in the EEPs, which were active on LM at 1%. However, flavonoids as well as aromatic acid esters were not detected in the EEPs, which were not active on LM at 1%. Chrysin, 4\',5-dihydroxy-7-methoxyflavanone and 3,4\',7-tri methoxyflavanone were the flavonoids detected in EEPs. Chrysin was detected in 12 EEPs out of 19, which were active on LM but not detected in the other 6 EEPs, which were not active on LM at 1%. The results of the study indicate that antibacterial activity of propolis is mainly depended on its source, chemical composition and the concentration of active compounds.


DETERMINATION OF TOTAL FLAVONOID CONTENTS IN PROPOLIS SAMPLES COLLECTED FROM EAST-ANATOLIA-TURKEY

Ömür Gençay¹*, Kadriye Sorkun¹, Bekir Salih<sup>2

1</sup> Department of Biology, Hacettepe University, Faculty of Science, 06532 Beytepe-Ankara-Turkey

² Department of Chemistry, Hacettepe University, Faculty of Science 06532 Beytepe-Ankara-Turkey

*corresponding author *gencay©hacettepe.edu.tr

Thirty propolis samples collected from 10 bee-farms located in the region of Kemaliye-Erzincan at east-Anatolia-Turkey were analyzed by UV-Visible Spectrophotometer and total content of flavonoids were determined after extracted into ethyl alcohol in saturated solutions. In this study, propolis samples were collected from every research beehive either in spring and summer times.

In this study, UV-Visible Spectrophotometer was used to determine the total flavonoid contents in the propolis samples measuring the adsorption peak areas. In UV-Visible Spectrophotometer, absorption peak in between 275-315 nm was characterize the total flavonoids in propolis samples. From the UV-Visible Spectrophotometric results, absorption areas representing total flavonoid contents were found to be between 237571500-2952480.

The highest adsorption area was obtained for the propolis sample collected from the 7th bee-farm (summer time collected sample), and the soluble organic components and flavonoid contents given absorption band in between 275-315 nm were found maximum. For this propolis sample, absorption peak area in between 275-315 nm was measured as 2375715000 absorption area. Around the 7th bee-farm, the population of Asteraceae, Fabaceae and Scrophulariaceae plants was very high. As a result, high flavonoid content was found to be highest in this propolis sample. The propolis sample collected from second bee-farm in summer time showed the lowest absorption peak area (2952480) in the same wavelength range. Around this bee-farm again, the same plants were found to be grown. But in this case, propolis sample was collected in summer time and we think that flavonoids are dominant in the early stage of the plants in spring time. The second highest flavonoid content was found in the propolis sample collected around 9th bee-farm and around this farm, the population of Salicaceae was very high. The dominant plant populations around the propolis samples having highest and lowest flavonoid contents (7th and 2nd bee-farms) were not different. But, the flavonoid content differences were resulted from the secondary plants and minor population type plants and also from the seasonal changes.


Rheological properties of selected Croatian honeys

Nela Nedic Tiban, Vlasta Pilizota, Drago Subaric, Djurdjica Ackar

Faculty of Food Technology, E-mail: nela.nedic©ptfos.hr

Studies on rheological behaviour of honey, like other fluid foods, could be important for applications related to handling, storage, processing, quality control, and sensory analysis of foods. The effect of temperature on rheological properties also needs to be documented because a wide range of temperatures encountered during processing and storage of liquid foods.

In this work, the rheological properties of four nectar varieties of Croatian honeys, among them three monofloral varieties (acacia, chestnut, sage) and meadow as polyfloral honey were investigated over a temperature range of 5 to 40 °C. The honeys had water content of 13.9 - 18.4 %. The rheological measurements of honey samples were carried out on rotational viscometer. The obtained results showed that all honeys exhibited Newtonian behaviour with viscosity reducing as the temperature was increased. Their viscosity varied according to the kind of honey and the temperature of measurement. The temperature dependence of viscosity was described using the Arrhenius equation. The activation energy ranged from 95.8 - 119.71 kJ/mol

Slovak centre of agricultural research Nitra, State veterinary and food institute Dolny Kubin

The quality and health safety of food are the complex of their characteristic properties defined in the legislative. Optimal level of the quality is often understood by consumers differently . The purpose of our works is to participate in protection of public health, as well as to study and to test substances which influence the biological value of food. Presented analyses don’t include all important parameters of quality and health safety of animal food of origin, but they compare achieved results from laboratory testing of selected Slovak and imported honey.

The quality of honey is affected by many different factors. They are removed from the honey bee colony until the honey is ready for human consumption.

The presence of adulterated honey in the Venezuelan market is not preventing its traditional use as a medicinal product of the hive. An effort to promote a local concern for honey quality control, was done last year, but only nine honey samples were received. For this reason, additional analysis were carried out to evaluate bioactive properties such as the phenolic content and the antioxidant capacity of honey from Anzoátegui, Barinas, Bolívar, Cojedes, Lara, Miranda and Trujillo. The total antioxidant activity (TAA) was measured as M Trolox equivalents, and the phenolic content was measured as mg gallic acid equivalents (GAE)/kg honey, compared with artificial honey made with fructose, glucose, maltose, sucrose and water. The TAA varied between 35.66-203.21 M Trolox equivalents and the phenolic content between 52.00-182.10 mg GAE/kg honey. Although the artificial honey control showed the lowest values for both measurements, of 17.39 M Trolox equivalents of TAA and 23.59 mg GAE/kg honey, the correlation between TAA and phenolic content of all honeys was low (r=0.52). Therefore, the antioxidant activity of this set of honeys is only partially explained by its phenolic content.

Twenty years ago beekeepers of the Venezuelan Andes in Mérida State were honey producers, but now pollen has become more rewarding because nectar flow has decreased. Brassica napus L. is abundant in the mountains and also the most frequently collected bee pollen, more than 50% of the bee pollen collected in Misintá Páramo along year 2005 was from B. napus. In this work, the concentrations of Pb and Hg were evaluated in fresh bee pollen of B. napus. The Pb analysis was done by electrothermal atomic absorption spectroscopy (ETAAS). The Hg analysis was done by flame atomic absorption spectroscopy (FAAS). A concentration of 268.31 ± 0.0008 mg Pb/kg bee pollen was found in fresh bee pollen of B. napus The method for the analysis of lead was validated by recovery studies, a value of 101.0 ± 4.8 % was obtained for lead. A concentration of 146.15 ± 0.0012 mg Hg/kg bee pollen was found in fresh bee pollen of B. napus. The method for the analysis of mercury was validated by recovery studies, a value of 96.0 ± 2.0 % was obtained for lead. For both measurements, the relative standard deviation (RSD%) was lower than 5%, indicating that the methods were exact, precise and free of interference. The concentration of the two heavy metals evaluated here, were safe for humans and lower than the Pb MRLs of 0.3 mg/kg fruit juices and1.0 mg/kg fruits and vegetables allowed by the Codex Alimentarius Commission.