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

E-mail: nkezic©agr.hr

With the diversity of plant species, The Republic of Croatia belongs to the richest countries in Europe. Known Croatian flora comprehends 8582 species; from that number 4266 are higher plant species dominantly from forest ecosystems. From known plant species 5,65% are endemic. Diverse climatic zones, which are characterized by three climatic types, continental, submediterranean and Mediterranean, contribute the most of all to the high biological diversity. In the Republic of Croatia forests cover 44% of land area and with forest lands, represent unique forest area on 2.485,611 ha. According to its structure, they are almost all natural. From the forest species, the most common are deciduous, with share of 84% in total timber stock, while conifer species are present on 16% of the total area. Forests are habitat for numerous honey producing plants, therefore in Croatia almost 70% of honey is produced from forest plants, from continental and Mediterranean area.

Share of forest produced honey is 31000 t with value of 34 million €, with growing demand on market. Specially important is honeydew honey from fir tree (Abies alba) and oak tree (Quercus sp.). Forest, due to specific structure and FSC management, are not loaded with pollution, honey from such areas has exceptional value.

Besides honey production, honeybees provide survival of numerous forest species, thus more hives can be found in forests.


Organic honey production and some quality parameters in Hungary

Dániel Szalai, Julianna Sütő, Enikő Szalai-Mátray

Szent Istvan University Doctoral School of Environmental Sciences, E-mail: szalai.daniel©kti.szie.hu

The Carpathian basin is a good source of the tasty liquid Robinia honey.

Hungary has some 200 thousand hectare of black-locust (Robinia pseudoacacia L.) forest. This gives the environmental basis for organic beekeeping as well. In the recent years the number of organic apiaries has increased though their group is still low compared to the conventional colleagues. Besides their relatively small group their product can be the leader of the very best quality honey on the market, therefore some basic components of these Hungarian honeys were investigated.

37 organic apiaries were selected from different parts of Hungary in 2004.

Honey samples (Robinia pseudoacacia, Asclepias syriaca and multifloral) were collected and analysed with HPLC to compare the fructose/glucose ratio, HMF content, and with classical analytical methods invertase, diastase activity and water content. The degree of crystallisation was calculated through visual and taste characteristics. Honeys originated from Black locust (Robinia pseudoacacia) showed the lowest deviation in F/G ratio, however other values may have been influenced by site specific and technological factors as well.

The studied Hungarian honeys showed 69.78% invert sugar in average. The average of fructose content was 41.61%, while the glucose detected 28.17%. The mean of HMF founded 5.67 ppm and the diastase activity was 17° Gothe.


Influence of the meteorology in the botanical characteristics of honey

M.Seijo, M. Chouza, F.J. Rodriguez-Rajo, J. Méndez*

Dept. Vegetal Biology and Soil Sciences. Faculty of Sciences. University de Vigo. Campus As Lagoas, 32004 Ourense., E-mail: mcoello©uvigo.es, Telf: +34 988387057, Fax: +34 988387001

Honey is a natural product directly derived from the territory. The biogeographical parameters (floral patterns, edafology, meteorology, etc.) are the main responsible for the characteristics that the honey will be present.

Our work group has a database with the pollen spectra of near 1500 honey sample from Galicia, produced between 1989 and 2005. The study of the pollen content indicates that the plants of main apicultural interest are: Castanea sativa, Eucalyptus globulus and Rubus sp.. A less importance have some like: Erica sp. (E. australis, E. umbellata, E. arborea), Trifolium, t. Campanula, etc..

The pollen of these plants appears in a common way in all the studied honeys. However, taking into account the interanual variability, important differences exist in the relative proportions of each one of them. Meteorology can be the principal cause of this variation due to there isn’t important differences in bee management.

With the purpose of to know if there is a pattern of the pollen spectra of honey related to the main bioclimatic index. We have calculated the principal bioclimatic index and have correlated them with the pollen spectra of the honey.

Research and Certification Center Federal, E-mail: microbio©tentorium.ru

The research has shown high natural level of microbe semination as a property of pollen load.

To determine the causes of high quantity of mold fungi and coliform bacteria in lots of pollen load (Khismatullin Rail G. and others, 2003) we conducted microbiological research of the samples gathered during May, June and July, 2005 in special nature reserve. Inoculation of the pollen load gathered by bees at forenoon was carried out next morning at 2-4°C. We analyzed 12 assays from 3 bee families and 36 samples of pollen load of different colors. General number of aerobic microorganisms was in the range of 6,6*103-1,5*107, moulds –3,0*103-6,6*105, yeast – up 7,5*105 CFU/g. Coliform bacteria ( mostly Enterobacter ssp. and Klebsiella oxitoca) are educed from 31 samples, E. coli - from 2 samples. Number of enterobacteria was 103-105 CFU/g. Lot by lot there was a growth of aerobic microorganisms and yeasts in number averaged for once selected assays of pollen load.

Date of taking a sample Number, CFU/g

Aerobes Yeast Moulds

24.05.05 1,8*105 3,2*103 1,2*105

07.06.05 6,1*105 8,5*103 6,2*104

21.06.05 2,3*106 - 2,4*105

28.06.05 2,5*106 1,0*104 8,7*104

13.07.05 6,6*106 1,2*105 1,6*105

E. coli and coliform bacteria Citrobacter and Enterobacter cloacae prevailed in all samples gathered in rural zones in summer 2004. Considering the data of common species of enterobacteria in commercial pollen load (Khismatullin Rail G. and others, 2005) we suppose the prevailing bacteria to be a result of influence of live-stock farming.

Slovak University of Agriculture in Nitra, E-mail: robert.chlebo©uniag.sk

Honey samples harvested between years 1962 and 2003 were analyzed for the amounts of humidity, saccharose, glucose, fructose, HMF, proline and diastase. The most important parameter for evaluation of honey aging is HMF. (Project VEGA 1/34/75/06)


Honey Discrimination of three Portuguese Beekeepers Associations by phenolic pattern

Luis G. Dias, Lillian Barros, Sonia Girante, Miguel VilasBoas, Letícia Estevinho

CIMO/ESAB, Campus Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal, E-mail: ldias©ipb.pt

Phenolic compounds (phenolic acids and flavonoids) in honey are known to have healthpromoting effects as antioxidants and anticarcinogens. These compounds, considered as the phytochemical profile, have also been used as biochemical markers for the honey geographic origin. Furthermore, these components make important contributions to organoleptic properties and to its physical and chemical properties.

This study involved honey samples of three nearby Beekeepers Associations of the Portuguese Trás-os-Montes region. The objective of this work was to improve the honey´s composition knowledge, with intention to know the differences between the honeys of Beekeepers Associations of this region and to gather information that might add economic value to this region?s honey.

The honey phenolic acids and flavonoids were extracted with Amberlite XAD-2 resin. Identification as well as quantification of these compounds was carried out via high performance liquid chromatography with Diode-Array detector.

The global phenolic pattern of honey contains protocatequic, p-hydroxibenzoic, caffeic, chlorogenic, vanilic, p-cumaric, elagic, and cinamic acids as well the naringenin, kaempferol, apigenin, pinocenbrin and crisin flavonoids. Using multivariate analysis, it is possible the honey discrimination of three Portuguese Beekeepers Associations by phenolic pattern.

CIMO/ESAB, Campus Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal, E-mail: ldias©ipb.pt

The antibiotics residual presence in honey is a current problem with negative implications, mainly commercial, since according to European legislation, antibiotic occurrence in honey samples is forbidden. In Portugal, there is a growing concern among Beekeepers with the residues of these products in honey and they are committed to producing and selling a clean health and natural product.

In order to overcome this concern, the Portuguese National Beekeepers Federation, the Beekeepers Associations and Bragança’s Agrarian Superior School participate in a project which aims to trace antibiotics in the honey and study the different ways of honey contamination by these residues (project financed by the Portuguese National Apicultural Program). The main objective is to trace the antibiotic residues to national level in order to infer the global situation of the Portuguese honey and to promote its quality; the second objective meets the necessities of the beekeeper of identifying sources of honey contamination.

The Beekeepers Associations send a number of samples proportional to the volume of honey production, with preference being given to the commercial honey with its own label. Different classes of antibiotics in honey were analyzed such as sulphonamides, tetracyclines and Streptomycin. The screenings were done by CHARM II and the positive results were confirmed by HPLC with fluorescence or UV detector. In the case of the positive results, inquires were sent to the Beekeeper Association so that a survey could be carried out, with the apiculture involved, for evaluation of the contamination origin.

The results show that clearly there are no residues of Streptomycin, while tetracyclines were found in only one sample. The sulphonamides are the main concern but, as the results are low, they reflect external or previous years contaminations.

Department of Human Nutrition, Slovak University of Agriculture in Nitra, Slovak Republic, katarina.sramkova©uniag.sk

Bee-collected pollen is an apicultural product which is composed of nutritionally valuable substances and contains considerable amounts substances, which may act as potent antioxidants. Carbohydrates are the principal components. Pollen is known to hold a wide variety of nutrients, including vitamins, minerals, amino acids, plant hormones, antioxidant factors, enzymes, phytosterols and more. The aim of the study was to measure antioxidant activity of bee-collected pollen samples from Papaver somniferum L. and analyse their chemical composition. There were selected components in pollen: proteins, various amino acids, fructose, and glucose. Absorbance at 470 nm was measured at different time intervals using spectrophotometer. Consequently antioxidant activity was calculated. Antioxidant activity of pollen from Papaver somniferum L. was 63.75  0.79 %. It was estimated also content of proteins in pollen (250.2 g.kg^-1). The amino acid content was - arginine 11.7 g.kg^-1, glycine 9.8 g.kg^-1, histidine 7.1 g.kg^-1, isoleucine 10.1 g.kg^-1, aspartic acid 18.7 g.kg^-1, glutamic acid 19.8 g.kg^-1, leucine 16.7 g.kg^-1, lyzine 16.0 g.kg^-1, phenylalanine 11.7 g.kg^-1, proline 20.3 g.kg^-1, serine 6.7 g.kg^-1, threonine 8.0 g.kg^-1, tyrosine 6.0 g.kg^-1, alanine 10.4 g.kg^-1, valine 12.4 g.kg^-1. The content of fructose was 188.5 g.kg^-1 and glucose 152.2 g.kg^-1. This work was supported by Science and Technology Assistance Agency under the contract No. APVT-20-026704.


Presence of antibiotics and sulfonamides in honey and royal jelly on the european market

Wim Reybroeck, Sigrid Ooghe, Els Daeseleire

Ministry of the Flemish Community, Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Unit, Brusselsesteenweg 370, B- 9090 Melle, Belgium, E-mail: wim.reybroeck©ilvo.vlaanderen.be

In 2005 in 5 European countries (Belgium, Great-Britain, Italy, Portugal and Spain) commercial honey samples were bought by different consumer’s organisations and screened for residues of streptomycins, tetracyclines, macrolides and chloramphenicol by means of accredited receptor and immunological tests. The screening of sulfonamides was performed with LC/MS-MS. Samples screened as positive were sent to an external lab for physicochemical confirmation by LC or LC-MS.

In 20.0 % of the honey samples at least one residue (streptomycin, tetracycline, tylosin, sulfathiazole and/or sulfamethazine) was present. All honey samples were free from chloramphenicol at the level of 0.1 µg/kg. For the 5 countries involved in the study, the following respective percentages of positive samples were obtained: 19.0% (BE, 4 out of 21), 15.0% (GB, 3 out of 20), 31.6% (IT, 6 out of 19), 35.0% (PT, 7 out of 20) and 0% (ES, 0 out of 20).

In 12 out of 23 royal jelly samples bought in Italy, Portugal or Spain high levels of chloramphenicol were found. The 4 samples of royal jelly bought in Belgium were free from residues of chloramphenicol. There was no sampling in Great-Britain.

A survey on portuguese honey physico-chemical parameters

H. Garção, S. Fonseca and M. Vilas-Boas

Centro de Investigação de Montanha, Escola Superior Agrária de Bragança, Campus de Santa Apolónia, 5300-955 Bragança, Portugal, E-mail: mvboas©ipb.pt

There is a growing demand of natural products in human diet, both due to the possible negative effects of synthetic food additives on human health and to the increased consumer perception of these products benefits. Among them we find honey, one of the most complex foodstuffs produced by nature and certainly the only sweetening agent that can be used by humans without processing. Honey characteristics are directly dependent on the flora visit by bees, and this is reflected on its composition. Physico-chemical analysis is used routinely to classify different types of honeys and evaluate their quality. With this work we present a general idea on the chemical properties of Portuguese honeys and contribute to their classification.

Honey samples were directly obtained from the beekeepers and collected at different locations across the country, including islands, with help of the National Federation of Portuguese Beekeepers and their partners. Standard parameters as colour, water content, pH, free acids, lactones, electrical conductivity and sugars, together with total phenols content, were collected using the methods validated by the International Honey Commission. Multivariate analysis helps to look at the sample in its entirety and not just at a single component, if we wish to untangle all the complicated interactions between the matrix constituents.

The results allowed us to associate parameters values to specific regions in Portugal, simply because there are differences in botanical origin: higher values of electrical conductivity are found for darker honeys and higher levels of total phenols and can be located mostly on the north/centre regions. South shows higher amounts of light honey with lower electrical conductivity.


DETERMINATION OF SULPHA DRUGS IN HONEY BY LC-MS/MS

Els Daeseleire and Wim Reybroeck

Ministry of the Flemish Community, Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Unit, Brusselsesteenweg 370, B- 9090 Melle, Belgium, E-mail: els.daeseleire©ilvo.vlaanderen.be

Sulpha drugs are sometimes used in apiculture to prevent or to treat bee diseases as nosemosis or bacterial brood diseases. This practice, causing residues in the honey is illegal in Europe since no MRLs for sulphonamides are fixed for honey (Council Regulation N°2377/90). In case of analysis, screening is done making use of the Charm II-Sulpha Honey test. For the confirmation of the results, a liquid chromatographic-tandem mass spectrometric method was developed and validated according to Commission Decision 2002/657. Clean-up was based on the method described by Maudens et al¹. In short, an aliquot of honey was dissolved in hydrochloric acid followed by an extraction with acetonitrile and a SPE clean-up making use of C₁₈ columns. The extracts were injected into the LC-MS/MS system. Sulfachoropyridazine was used as internal standard. For the 7 sulpha drugs tested in honey the decision limit (CCα) was 2 µg/kg and depending on the type of sulpha drug the detection capability (CC) was in the range of 2.2 – 2.4 µg/kg. All these values are far below the action limit of 20 µg/kg set in Belgium for the group of sulphonamides. Other validation parameters tested were linearity, specificity, recovery, repeatability and intra-reproducibility.

¹Maudens K.E. and Lambert W.E., Proceedings volume 2, Euro Food Chem XII, Strategies for safe food, Brugge, 24-26 september 2003


Pollen content of summer honeys from south-eastern Poland

Ernest Stawiarz, Katarzyna Grzechnik, Anna Wróblewska

Botany Department, Agricultural University, Akademicka 15, 20-950 Lublin, Poland,

In the years 2001-2005 the samples of twenty nine summer honeys were collected from apiaries of different localities of south-eastern Poland. Pollen analysis of honey sediments was made according to the methods recommended by the International Commission for Bee Botany IUSB (Louveaux et al. 1978), whereas palynological examination by Zander classification (1935, 1937).

Results of microscopic studies have shown the presence of 82 pollen taxa (from 42 plant families), among which 61 represented by nectariferous and 21 by non-nectariferous – anemophilous and entomophilous plants. Non-nectariferous species pollen percentage in the honey samples hesitated between 0.6 and 66.4. A particular honey contained in total 13-41 (averaging 27) pollen types. The honeydew elements have been found in 22 samples.

The most important source of nectar for bees were the following taxa: Brassicaceae and Trifolium repens (in 100% of samples), Prunus, Rubus (93.1%), Centaurea cyanus, Salix (86.2%), Tilia, Anthriscus (82.8%), Fagopyrum (79.3%), Taraxacum (75.9%), Frangula (72.4%). Pollen of the mentioned taxa appeared in the investigated samples as a dominant, secondary or important minor. Among the nectarless taxa the highest frequency (over 50%) reached: Poaceae, Rumex, Plantago, Ranunculus, Filipendula.

Eight of the examined honeys were unifloral: three from Fagopyrum and one by one from Cucurbita, Myosotis, Prunus, Robinia and Tilia. The remaining 21 samples were classified as multifloral honeys with high pollen contribution of Brassica napus and other Brassicaceae, Fagopyrum, Phacelia, Rubus and Trifolium repens.


Quantitative palynological analysis of honey: an automatic method much faster and accurate

M.Scala, G. Aronne

Palynological Analyses Centre (CeAP). University of Naples Federico II. Faculty of Agricolture, via Università, 100 – 80055 – Portici (Naples), Italy, E-mail: micscala©unina.it

Palynological analysis of honey is important not only for the determination and control of the geographical and botanical origin of honey but also for obtain further information about honey extraction and conservation. These tests consist in a quantitative and a qualitative analysis. The first gives information on the total number of figurative element present in the honey. The second achieves the taxonomical classification and the percentage of different pollen types occurring in the samples. Both analyses are time consuming.

In this work we present a tool for the automatic quantitative palynological analysis of honey. For the preparation of the slide we use the procedure described in Von der Hoe et al., 2005. Apidologie (35) S18-S35. Slides are observed under an epifluorescence microscope. An appropriate combination of the excitation, dichromatic and emission filters allows the rapid distinction of the figurative elements present in the honey. The subsequent count of the plant elements is made by a dedicated software for image analysis named Pollen^® developed from this group. We also demonstrated that this method is more faster and accurate: only few minutes are needed to count up to 5000 figurative elements.

Turkey has enormous beekeeping potential with over 4.393 million bee colonies and diverse floral ecotypes. In terms of honey production Turkey has been ranked 2nd in the world rank with 73.929 tone produced in 2004.

Many various honeys (mono floral and multi floral) are produced in Turkey. Also countless various plant species raised in the country. The most important honey sources are pine, citrus, sunflower, acacia, linden and compound honey.

Several different criteria are commonly used in evaluating the quality of honey in international trade. These quality criteria include sensorial characteristics (i.e., flavor, color and taste) and biochemical analysis; moisture, pH, activity of hydrogen peroxide, invert sugar, HMF content, diastase activity, mellisopollinolgical analysis. In addition the extensive analysis such as prolin content, rate of K/Na, polarimetric analysis and C 13 test are done for adulterated honey.

In this study a total of Twenty Indian honey samples were analyzed and botanically characterized: these included sunflower (Helianthus annuus L.), mustard, toria, sarson, raya (Brassica sp), eucalyptus (Eucalyptus lanceolatus L.), berseem clover (Trifolium alexandrium L.). Unifloral honey was considered as such whenever the dominant pollen was found at over 45% of total pollen. The samples have been analyzed to determine: moisture, pH, hydroxymethylfurfural, diastase activity, optical rotation, conductivity and color variables (L, a, b,). A Brookfield LV model (LV, Spindle31) was used to measure rheological properties of honey samples. All of these honey varieties from different floral sources exhibited Newtonian behaviors. The apparent viscosity was found to decrease with the temperature and temperature dependence of viscosity was contrasted versus Arrhenius model (μ= μo e Ea /RT). The temperature effect on the viscosity followed an Arrhenius- type relationship. The activation energy ranged from 72930.40 to 87105.77 J/Kg mol.

CIMO/ESAB, Campus Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal

Phenolic compounds (phenolic acids and flavonoids) in honey are known to have healthpromoting effects as antioxidants and anticarcinogens. These compounds, considered as the phytochemical profile, have also been used as biochemical markers for the honey geographic origin. Furthermore, these components make important contributions to organoleptic properties and to its physical and chemical properties.

This study involved honey samples of three nearby Beekeepers Associations of the Portuguese Trás-os-Montes region. The objective of this work was to improve the honey’s composition knowledge, with intention to know the differences between the honeys of Beekeepers Associations of this region and to gather information that might add economic value to this region’s honey.

The honey phenolic acids and flavonoids were extracted with Amberlite XAD-2 resin. Identification as well as quantification of these compounds was carried out via high performance liquid chromatography with Diode-Array detector.

The global phenolic pattern of honey contains protocatequic, p-hydroxibenzoic, caffeic, chlorogenic, vanilic, p-cumaric, elagic, and cinamic acids as well the naringenin, kaempferol, apigenin, pinocenbrin and crisin flavonoids. Using multivariate analysis, it´s possible the honey discrimination of three Portuguese Beekeepers Associations by phenolic pattern.

Faculty of Food Technology and Biotechnology, E-mail: dmatkov©pbf.hr

Honey samples that are available commercially, differ in quality on account of various factors. These include geographical, seasonal and processing conditions, floral source, packaging and storage period. Although Croatia is a small country, there’s a wide range of different types of honey present on domestic market. In this study a total of 220 Croatian honey samples of known origin harvested in three consecutive years (2003., 2004. and 2005.) were analysed. Among honeys studied there were: 108 samples of acacia, 21 floral, 18 sage, 27 chestnut and 46 meadow. The following parameters were determined: water, total reducing sugars, sucrose, electrical conductivity, acidity, hydroxymethylfurfural (HMF) content, diastase activity and proline content. The aim was to evaluate the quality of selected honeys according to the current legal regulations in Croatia.


UPLC-TOF-MS – a useful tool for the analysis of honey?

S. Trautvetter, I. Kölling-Speer, K. Speer

Prof. Dr. Karl Speer, Institute of Food Chemistry, Technische Universität Dresden, Bergstraße 66, D-01069 Dresden, Germany, E-mail: sophie.trautvetter©chemie.tu-dresden.de

The authenticity of honey is almost exclusively characterised by the microscopic analysis of pollen which can only be carried out by trained personnel. Therefore, chemical-analytical indicators are required to determine the individual kinds of honey.

Herefore, as already published [1,2,3], phenolic acids and flavonoids are suitable substances.

Most of the phenolic acids can easily be determined by GC-MS after derivatization, but HPLC-DAD would be preferable for analysing both groups. Nevertheless, due to the quantity of the compounds in honey, their similar structures and their different UV-sensitivity, the analysis is a complicated matter.

Recently, the Ultra Performance Liquid Chromatography combined with TOF–MS (UPLC-TOF) was used by our work group. The advantages and disadvantages will be presented and discussed.

Literature:

[1] Steeg, E., Montag, A.; Z Lebensm Unters Forsch 1988, 187, 115-120

[2] Tomás-Barberán, F. A., Martos, I., Ferreres, F., Radovic, B.S., Anklam, E.; J. Sci. Food Agric. 2001, 81, 485-496

[3] Yao, L., Jiang, Y., Singanusong, R., Datta, N., Raymont, K.; Food Res Int 2005, 38, 651-658

Recently, the process of ultrafiltration was applied to honeys to avoid crystallization. Thereby, pollen and antibiotics as well as larger molecules like proteins can be removed [1].

The effects of ultrafiltration on the composition of honey, especially on the enzymes diastase and saccharase, on chloramphenicol and HMF will be introduced. The ultrafiltration process was carried out on the laboratory scale, and the products were analysed and compared. The results presented here show that the contents of these components are considerably influenced by ultrafiltration.

Literature:

[1] Marshall, A., The Middlesex Bee, 2004, 15 (2), 1

Influence of ripening honeys in controlled condition on their physicochemical property

Piotr Semkiw, Wojciech Skowronek, Piotr Skubida, Dariusz Teper

Research Institute of Pomology and Floriculture, Apiculture Division Pulawy.Poland

Samples of honey harvested before the end of a nectar flow. Several honey combs with uncapped honey were extracted and dehydrated in a chamber equipped with a dehumidifier. Before and after the dehydrating process the samples of honey were taken to be analysed. Other honey combs were left in the hive and samples of ripen honey (conventional) were taken after the end of the nectar flow to compare the quality of undehydrated, dehydrated in controlled conditions and conventional honey. Water content, free acidity, sugar content, electrical conductivity, diastase activity were determined in the samples. The initial content of water in honeys was 23.06% and after 12 hours of the dehydrating process honey humidity dropped by 1.78% on average. After 36 hours of the dehumidification process the average content of water was 16.46%. In conventional honeys the average content of water was 17.91% which is higher than that in honey dehydrated in controlled conditions.

The reduction of excess water content caused increase of free acidity, sugar content and diastase activity. Diastase activity increased average by 3.49 on Schade scale. Free acidity content increased by 1,1 mval/kg in comparison to undehydrated honey. Sugar content also increased; the sum of fructose and glucose increased by 5.41% on average and sucrose by about 0.22%. A little higher average diastase activity and free acidity were determined, in comparison to dehydrated honey but sugar content was lower. The ripening of honey in controlled conditions allows the water content in honey to decrease and it is not harmful to honey quality. Reduction of water content causes increase of free acidity, sugar content and diastase activity. The parameters of dehydrated honey do not differ considerably from those of conventional honeys.


Pollen spectrum and physicochemical attributes of Croatian Mediterranean multiforal honeys

Ljiljana Primorac¹, Dragan Bubalo², Daniela Kenjerić ¹, Antonija Perl-Pirički¹, Ivana Flanjak ¹, Milena L. Mandić <sup>1

1</sup>.J.J. Strossmayer University of Osijek, Faculty of Food Technology, F. Kuhača 18, HR-31000 Osijek, Croatia

².University of Zagreb, Faculty of Agriculture, Svetošimunska 25, 10000 Zagreb, Croatia

The aim of this work was to determine which floral sources prevail in Croatian Mediterranean multifloral honeys and its physicochemical characteristics. Honey (55 samples) verified by pollen analysis as multifloral, were collected during the 2004 harvest directly from beekeepers from different locations across Croatian Mediterranean area. The following determinations were carried out: moisture content, electrical conductivity, specific rotation, pH and acidity, hydroxymethylfurfural (HMF), diastase and invertase activity, proline content and sugar composition.

Pollen analysis of honey samples showed a wide variety of botanical sources. In a melissopalynological study of investigated honey samples, 78 pollen types were identified. Samples ranged from 8 to 21 different plant species. The principal species identified were: Paliurus spina christi, Fraxinus ornus and Trifolium pratense gr.

In accordance with a variety of nectar sources, samples have relatively wide range of physicochemical parameters: electrical conductivity (0.14 mS/cm - 0.87 mS/cm), pH (3.74 - 5.12) and free acidity (11.3 meq/kg - 37.1 meq/kg), specific rotation ((-15.4) - (-4.1)), proline content (234 mg/kg - 960 mg/kg), invertase (6.2 U/kg - 405.8 U/kg) and diastase activity (Schade scale) (14.1- 57.7).

Though samples are diverse, all together they have high enzymatic activity which is followed by low HMF content.

Screening of the potential presence of pyrrolizidine alkaloids in honeys by spectrophotometric determination

R.A.Pérez Martín, M. González Lorente, P. Lorenzo Lozano, and C. de Lorenzo Carretero

Dpto. de Agroalimentación. Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA). Finca El Encín, Aptdo. 127, 28800 Alcalá de Henares, Madrid (Spain).

Pyrrolizidine alkaloids (1,2-dehydro-pyrrolizidine ester alkaloids, PAs) are a large group of natural toxic products from the secondary metabolism of some plants. Plants containing these alkaloids are found in representatives of the families Fabaceae (Leguminoseae), Boraginaceae and Asteraceae (Compositae). The genus Echium (Boraginaceae) is widely distributed across Europe and their species Echium plantagineum L. and Echium vulgare (Viper’s bugloss) are important nectar sources for honeybee. Honey research, as potential way for the APs ingest, is relatively recent and complex.

Melissopalynological analysis of 127 Spanish honeys was carried out, the results showing an important incidence of the genus Echium in these samples, thus suggesting the necessity to develop a method for the screening of the samples. With this objective, different extraction procedures were assayed using honey free of Echium palynomorphs, fortified with two PAs standards. The extractive method selected for further analysis was the SPE with Extrelut column (Merck).

Screening of honey extracts were done by means of spectrophotometric analysis of the coloured solution resulting of a first treatment of the extract, to yield a pyrrole derivative by PA’ dehydrogenation, followed by coupling with 4-dimethylaminobenzaldehyde to obtain the coloured solution. In this screening, twenty one honeys with different quantitative presence of Echium pollen were spectrophotometrically evaluated, the results yielding positive identification of alkaloids in honeys with some content of pollen from Echium in their melissopalynological analysis. The effect of the matrix colour was taken in account in these determinations.

Spectrum of meliferous plants exploited by Apis mellifera adansonii in highlight soudano- guinéenne zone of West Cameroon

Dongock Nguemo Delphine

University of Dschang, E-mail: dndongock©yahoo.fr

Melissopalynology analyses of 30 honey samples collected between September 2002 and March 2003 in the westren soudano-guinean highlands of Cameroon were carried out in order to evaluate frequency and intensity of exploitation of melliferous plants by Apis mellifera adansonii The main result were as followed:

- Spectrum of melliferous plants gathered is wide and diverse. Indeed pollen of 88 genus of melliferous plants of 46 families were identified in honey samples. In term of number of plants genus the must represented were Asteraceae, Euphorbiaceae an Myrtaceae.

- As concern frequency of exploitation of melliferous plants, melliferous plants could be grouped into three categories: must frequently gathered melliferous plants : Eucalyptus, Coffea, Bidens and Terminalia; moderately gathered melliferous plants : Elaeis, Phoenix, Lannea and Julbernardia. Finally less frequently gathered melliferous plants with their pollen present in less than 30% of honey samples (Salix, Casuarina, Pterocarpus etc)

- When intensity of exploitation is considered, the must intensively exploited melliferous plants by Apis mellifera Adansonii represent 22,7% of genus grouped into 16 families. Pollen of 8,0% melliferous taxa gathered belonging to six families appeared as dominant in honey samples. More than two third (92,0%) of the melliferous plants appeared as accessory, secondary or rare pollen. Eucalyptus was the only melliferous plants whose pollens were altogether as dominant, accessory and rare in honey samples.

From their pollinic composition, 60% of honey samples were unifloral with Eucalyptus and Helycrisum pollen dominating, against 40% which were multifloral honey.


UNIFLORAL HONEY CHARACTERIZATION ATTEMPT BASED ON QUALITY CONTROL PARAMETERS

M.T.Sancho¹*, M.A.Fernández-Muiño¹, M.M.Cavia¹, S.R.Alonso-Torre¹, G. Moreno¹, P. Rodríguez¹, I.Mato², S.Suárez-Luque², M.P.Sánchez², J.F. Huidobro^2
1 Universidad de Burgos. Departamento de Biotecnologia y Ciencia de los Alimentos. Area de Nutricion y Bromatologia. Facultad de Ciencias. Plaza de Misael Banuelos Garcia s/n. 09001 BURGOS (Castilla y Leon). SPAIN.

² Universidad de Santiago de Compostela Departamento de Química Analítica, Nutrición y Bromatología. Facultad de Farmacia. SANTIAGO DE COMPOSTELA (Galicia) SPAIN.

Unifloral honey characterization is usually based on melissopalynology, organoleptic characteristics and very specific parameters such as amino acids, flavors, minerals, and flavonoids, among others. These parameters are difficult to determine within small and local laboratories, in which only routine quality control parameters are completely standardized. The groundwork of this work has been to try to characterize five types of Spaniard monofloral honeys by determining electrical conductivity, free acid, formol number, proline content and diastase, invertase and ß-glucosidase activities. 107 samples, harvested in 2001, 2002 and 2003 were analyzed: 22 eucalyptus (Eucalyptus sp.) honeys, 35 rosemary (Rosmarinus officinalis) honeys, 15 citrus blossom (Citrus sp.) honeys, 33 heather (Ericaceae) honeys and 2 honeydew honeys. Depending on the color and botanical origin of the samples, the results have been different. Dark honeys (heather and honeydew) have shown the highest values for all the parameters analyzed, in contrast with light honeys (citrus and rosemary) that have shown the lowest ones. In general, when comparing heather and honeydew honeys, proline and ß-glucosidase activities mean values have been higher in heather honeys; electrical conductivity average has been slightly higher in heather honeys, free acid mean values have been similar in both heather and honeydew honeys and formol number, diastase and invertase averages have been higher in honeydew honeys. When comparing citrus and rosemary honeys, electrical conductivity, formol number, proline, diastase, invertase and ß-glucosidase activities mean values have been slightly lower in citrus honeys, and free acid has been similar in both citrus and rosemary honeys. Despite the fact that light honeys have not been botanically characterized with the parameters analyzed, statistical dicriminant analysis has correctly classified 95.5% eucalyptus honeys, 80% heather honeys and 100% honeydew honeys, which means that with simple, rapid, and low cost routine quality control parameters, it is possible to have an idea of the botanical origin of dark honeys.

NOTE: The Spaniard INIA (Ministerio de Ciencia y Tecnología, Spain) has supported this work under the project CAL01-066-C7-3.


GLUCOSE-OXIDASE AND CATALASE ACTIVITIES OF SPANISH UNHEATED HONEYS

M.A.Fernández-Muiño¹*, M.T. Sancho¹, M.M. Cavia¹, S.R. Alonso-Torre¹, G.Moreno¹, P. Rodríguez¹, M.P.Sánchez², I. Mato², S.Suárez-Luque², J.F.Huidobro^2
1 Universidad de Burgos. Departamento de Biotecnologia y Ciencia de los Alimentos. Area de Nutricion y Bromatologia. Facultad de Ciencias. Plaza de Misael Banuelos Garcia s/n. 09001 BURGOS (Castilla y Leon). SPAIN.

² Universidad de Santiago de Compostela Departamento de Química Analítica, Nutrición y Bromatología. Facultad de Farmacia. SANTIAGO DE COMPOSTELA (Galicia) SPAIN.

Glucose-oxidase and catalase are enzymes that have been hardly studied in honeys. Analysis of both enzymes is not simple, because there are lots of interferences due to many components of this food. However, the determination of glucose-oxidase and catalase is very important because these enzymes are directly and inversely correlated with antibacterial activity of honey, respectively. Within honey, bees’ glucose-oxidase produces hydrogen peroxide which is considered by many researchers as one of the main antibacterial honey agent. Furthermore, glucose-oxidase contributes to honey acidity due to the formation of gluconic acid what makes honey more stable against fermentation. The origin of catalase is mainly vegetal, although it can be also produced by some yeasts and other microorganisms. As catalase breaks hydrogen peroxide molecule, its activity is inversely correlated with antibacterial activity of honey. Glucose-oxidase has been determined by spectrophotometric measurement, at 400 nm of the product of the reaction between o-dianisidine, peroxidase and hydrogen peroxide obtained from the action of glucose-oxidase on glucose. Catalase determination is based on the reaction of this enzyme with an excess of hydrogen peroxide with the subsequent spectrophotomeric measurement at 400 nm, of the reaction product between the reminder hydrogen peroxide, o-dianisidine and peroxidase. For the analysis of both enzymes a previous dialysis step is necessary to remove interferences. Glucose-oxidase and catalase have been determined on 23 Spanish unheated honeys. 10 samples were Rosmarinus sp. honeys. 6 samples were Ericaceae honeys. 2 samples were Citrus sp. honeys. 2 samples were Eucalyptus sp. honeys. 2 samples were polyfloral honeys and 1 sample was honeydew honey. Mean value for glucose-oxidase was 305.7 μg H2O2/g honey/h, with values ranging from 106.5 to 718.5 μg H2O2/g honey/h. For catalase, values ranged from 1.6 to 107.6 Kf 10-3/g honey/min, with an average of 41.2 Kf 10-3/g honey/min.

NOTE: The Spaniard INIA (Ministerio de Ciencia y Tecnología, Spain) has supported this work under the project CAL01-066-C7-3.


A comparison of pine honey’s volatile compounds with those of honeydew produced from Marchalina hellenica

Chrisoula Tananaki, Andreas Thrasyvoulou

Laboratory of Apiculture-Sericulture, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, E-mail: txrysa©yahoo.com

Honey is derived from flower nectar and honeydew. Pine honey is the most common Greek honeydew honey, which is produced in autumn from honeydew secreted by the insect Marchalina hellenica. This insect is restricted to Pinus brutia, Pinus halepensis, Pinus sylvestris, and Pinus pine.

In this work pine honeys samples and honeydew secretions from Marchalina hellenica of the same area were collected and analysed using a Purge & Trap – GC – MS. A total of 46 and 27 volatile compounds were identified in the samples of pine honey and honeydew respectively. A great proportion of the volatile compounds in honeydew were terpenes. The substances α- and β-pinene (31.3 and 17,7%), δ-3-carene (30,9%), α-terpinolene (4,4%), camphene (4,3%) and β-phellandrene (3.2%) were found in high percentages. Fourteen of honeydew’s volatile compounds were also detected in the honey. The results of this research verified that some of the volatile compounds of tree are transferred to the honey without convertion.

Laboratory of Apiculture-Sericulture, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, E-mail: txrysa©yahoo.com

Honey is a concentrated solution of various sugars, which is produced by bees from the nectar and honeydew. The 5% of the annual greek honey production is fir honey. The heating process effects its quality as changes observed in moisture, sugars, amino acids, enzymes, HMF microorganisms and volatile compounds.

In the present study fir honey samples were heated in different temperatures for four period of time and the samples were analysed using a Purge & Trap – GC – MS system. Seven furan derivates (2,3-dihydro-4-methyl-furan, dihydro-methyl-3[2H]-furanone, 2-furanyl-methanol, 5-methyl-2-furanyl-carboxaldehyde, benzofuran, 2,2’-furan, 2-ethyl-5-methyl-furan) were formed during heating. Furfural and 1-(2-furanyl)-ethanone were detected in the control honey but their concentrations increased particularly after heating at 65˚C. The formation depended from the temperature and the duration of the heating.

Department of Animal Science, Agriculture Faculty, University of Mustafa Kemal, Hatay/Turkey

Classification is very important for the evaluation of agricultural crops. Unfortunately, subjectivity, tediousness and inconsistency in grading by human experts enforce the post harvest process to apply automation in honey grading. Fuzzy logic (FL) was applied as a decision making support to grade honey in this study. Chemical features such as the mineral (%), Invert sugar (%) and Moisture content (%), Acidity (meq kg-1), HMF (mg kg-1) Diastase level, and Sucrose (%) were measured through different laboratory equipment and methods. The same honey samples were graded by both a human expert and a FL system designed for this purpose. Grading results obtained from FL showed 97,14 % general agreement with the results from the human expert, providing a safe check for expert’s observations in honey grading.

Kayseri,Turkey.

A survey of 25 honey samples of different botanical origin, collected all over the Turkey was carried out in order to asses their heavy metal contents. The aim of this study was to determine the levels of cadmium (Cd), lead (Pb), iron (Fe), manganese (Mn), copper (Cu), nickel (Ni), chromium (Cr) zinc (Zn) aluminum (Al) and selenium (Se) in honey samples from different region of Turkey. Heavy metal contents were determined by flame and graphite furnace atomic absorption spectrometry after microwave digestion, wet digestion and dry ashing. The accuracy of the method was corrected by standard reference material (NIST-SRM 1515 Apple leaves). The contents of trace heavy metals in honey samples were found to be in the range of 0.23-2.41 mgg^-1, 0.32-4.56 mgg^-1, 1.1-12.7 mgg^-1, 2.1-10.2 mgg^-1, 9.6-105.8 mgkg^-1 and 2.6-29.9 mgkg^-1, 2.4-37.9 mgkg^-1, 1.1-17.9 mgkg^-1, 83-325 mgkg^-1, 38-113 mgkg^-1 for Cu, Mn, Zn, Fe, Pb, Ni, Cr, Cd, Al and Se respectively. Aluminum was the most abundant of the element while iron was the least present element in Turkish honeys.

¹ Ege University, Faculty of Agriculture Department of Animal Science, 35100-Bornova-Izmir/TURKEY, e-mail: banu.yucel©ege.edu.tr ² Doga Beekeeping Company, Tekirdag / TURKEY³ Mustafa Kemal University, Faculty of Agriculture, Dept. of Animal Science, Antakya/TURKEY

Turkey is the biggest pine honeydew honey producer and exporter in the world. 92% of world’s pine honeydew honey production is realized in Mugla Region of Turkey. The economical importance of pine honeydew honey is quite significant. Recent years, the antibiotic residue arguments are arised in pine honeydew honey from Turkey and exportation was decreased. The aim of this study was to explain the last datas (year 2005) of residue of pine honeydew honey from Turkey..

The eighty-two pine honeydew honey samples were harvested from Mugla Region of the country. Samples were analysed for the presence of residues of sulfonamide and tetracyclines in Ege University Center for Drug R&D and Pharmacokinetic Applications, in 2005. HPLC analytical method was used for the determination of antibiotic residues in honey. Residues of veterinary drugs were found in a limited number of honey samples, namely; tetracycline (1 sample of chlortetracycline, 1 sample of doxycycline, 3 samples of oxytetracyclin) 5 out of 82 ( 6 %) samples and sulfanamid (8 samples of sulfamethazin, 3 samples of sulfamethizol, 2 samples of sulfamethoxyprazin) 13 of 82 (16 %) samples were found. In comparing with last two years, the sharp decrease seems of the honey contamination with antibiotic residues but not the disappearence of the problem. Turkish pine honeydew honey will take a better place in the European market in near future.

University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

E-mail: urska.dobersek©bf.uni-lj.si

Specific rotation (SR) depends on the ratio among glucose and fructose content. Nectar types of honey have negative SR, because fructose as a prevalent sugar is laevorotatory, while honeydew types have positive SR, because glucose as a prevalent sugar is dextrorotatory. In this way it is possible to discriminate between nectar and honeydew as the main honey sources. European Directive concerning honey differentiates nectar and honeydew honeys with electrical conductivity (EC), where 0.8 mS/cm is set as the limit value; in pure nectar honey types must be below it, and in honeydew types must be above it. Lime and chestnut honeys that are of both, nectar and honeydew origin, are exceptions in this limitation. The results of our research proved that even SR can not be used in the case of these two honeys. Nevertheless, the combination of the EC and SR is a great tool for discrimination of this two specific types of honey from all other types and mixtures, since lime honey has EC below 0.8 mS/cm and positive SR (1st quadrant), and chestnut honey has EC above 0.8 mS/cm and negative SR (3rd quadrant), respectively. For comparison, acacia and multifloral honeys have EC below 0.8 mS/cm and negative SR (4th quadrant), and honeydew honeys have EC above 0.8 mS/cm and positive SR (2nd quadrant). The linear correlation between EC and SR for acacia, multifloral and honeydew honeys is statistically significant at the 0.01 level, it is very strong (R2 = 0.96) and equal to: SR = 41.3 cm/mS * EC – 34.3. On the contrary, the correlation for lime and chestnut honey does not exist.

Department of Food Science and Technology, Biotechnical Faculty,

University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

E-mail address: jasna.bertoncelj©bf.uni-lj.si

The color of five types of Slovenian honeys, namely acacia, lime, chestnut, multifloral and honeydew honey (ten samples of each) was investigated by means of two methods: color intensity (ABS450) and determination of color parameters through the CIE L*a*b* tristimulus method. In addition electrical conductivity, ash content, proteins and total phenolic content of honeys were determined and the correlation coefficients with the color were calculated.

The color of honey is related to the content of minerals, plant pigments, pollen and phenolics and is characteristic for honey floral origin. The results of this study demonstrated that the color of Slovenian honeys is very different and varies from almost colorless to dark brown. The brightest are acacia and lime honeys, almost colorless to pale yellow and white to cream or ivory, with yellow or green shade, respectively. Multiflower honeys are very colorful from yellow to brown depending on plant source and honeydew presence. The color of chestnut honey is amber, more or less dark, with reddish shade, while honeydew honeys are light to dark brown, with reddish or green shade.

Electrical conductivity and the contents of ash, proteins and total phenolics of the studied honey samples differ widely. The highest values of these parameters were obtained for chestnut and honeydew honey and the lowest for acacia honey. The correlation matrix showed a significant correlation between honey color and all other physicochemical parameters analyzed.

The effect of the drying process on the antioxidants vitamins from bee pollen

K.C.L.S. Oliveira, M. Morya, R.A.B. Azedo, E.W. Teixeira, M.L.T.M.F. Alves,
A .C.C.C. Moreti, L.B. Almeida-Muradian

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

Bee-collected pollen (bee pollen) is consumed like a human dietary supplement but such as other protein and moisture rich foods, it can lose its nutritional value when stored or processed incorrectly. To avoid bee pollen deterioration it must be dried quickly and there are many preserving methods as follow: open air drying, artificial heating, solar drying and silica gel treatment. For commercial scale production, the most commonly method used is the artificial heating to achieve moisture of 4% (established by the Brazilian regulation). It can be obtained by raising the temperature to 40-42ºC. Unfortunately, in some cases, this temperature tends to diminish the vitamin content. The aim of this study is to quantify the antioxidant vitamins: C, E and beta-carotene as provitamin A in fresh and processed samples of bee pollen and to evaluate the effect of two drying processes in the vitamin content. Ten samples of fresh bee pollen were dried by either conventional method (drying at 42ºC) or by an alternative method (drying at 30-35ºC). Vitamin C was quantified by potentiometric titration, vitamin E by HPLC-normal phase and beta-carotene by open column chromatography. In fresh samples, vitamin content varied between 13.5 and 42.5 ug/g for vitamin E, 56.3 and 198.9 (ug/g) for beta-carotene and 273.9 and 560.3 ug/g for vitamin C. For the drying process at 42ºC it was observed a loose of the vitamins as follows: 35.5 % (vitamin E), 56.5 % (provitamin A) and 8.1 % (vitamin C). For the drying process at 30-35º C it was observed a lost of the vitamins as follows: 28.9 % (vitamin E), 46.4 % (provitamin A) and 4.0 % (vitamin C). Both methods were efficient in drying the bee pollen but the alternative method, which uses the lower temperature, was more efficient in retaining the vitamins.
Acknowledgment: FAPESP and CNPq


Evaluation of nectar flow in new low erucic acid and low glucosinolate content lines of Sinapis alba, Brassicaceae

Marzena Masierowska¹ and Teresa Piętka<sup>2

1</sup>Dept. of Botany, Agricultural University, Lublin, ²IHAR, Poznań, Poland.

Flowers of Sinapis alba show typical entomophilic features. They offer both nectar and pollen to pollinators. Sinapis alba is known as a good melliferous crop, eagerly visited by honey bees. Plants are cultivated mainly for seeds, which set highly depends on the presence of insects. After an improvement of chemical composition of seeds, S. alba can be an alternative oilseed crop, replacement for canola crops damaged by winter frosts and a source of high protein supplements.An important research objective at the IHAR, Poznań, Poland is the development of low erucic acid and low glucosinolate content lines, resulting in plants, which are devoid of sinalbin. In 2005 a study on a beekeeping value of the best 36 lines has been started. During the peak of flowering the abundance of nectar secretion as well as sugar productivity were investigated. Moreover, flowers morphology was examined.In flowers of all studied lines nectar is secreted by the median and lateral pair of nectaries, situated in the region of filament bases. However, differences were found in morphology and activity of these pairs. Usually, the lateral glands produced more nectar than the median ones, but in 7 lines the activity of both pairs was equal. In florets of 3 lines the stamens were fused, making an access to nectar difficult. Mean nectar productivity per 10 flowers differed significantly between the lines and ranged between 3.11 mg and 18.67 mg. Mean concentration of nectar was 12% - 40.3%. Ten flowers of the observed lines secreted from 0.7 mg to 4.79 mg of sugars in nectar, on average. These differences in nectar productivity and accessibility between the breeding lines can influence insect visitation on their flowers and then the yield of seeds.