Palynological evaluation of selected honeys from Romania

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1 Grana ISSN: (Print) (Online) Journal homepage: Palynological evaluation of selected honeys from Romania Irina Dobre, Petru Alexe, Olga Escuredo & Carmen Maria Seijo To cite this article: Irina Dobre, Petru Alexe, Olga Escuredo & Carmen Maria Seijo (2013) Palynological evaluation of selected honeys from Romania, Grana, 52:2, , DOI: / To link to this article: Published online: 31 Oct Submit your article to this journal Article views: 365 View related articles Citing articles: 7 View citing articles Full Terms & Conditions of access and use can be found at

2 Grana, 2013 Vol. 52, No. 2, , Palynological evaluation of selected honeys from Romania IRINA DOBRE 1, PETRU ALEXE 1, OLGA ESCUREDO 2 & CARMEN MARIA SEIJO 2 1 Department of Biochemistry, Dunarea de Jos University, Faculty of Food Science and Engineering, Galati, Romania, 2 Department of Vegetal Biology and Soil Sciences, University of Vigo, Faculty of Sciences, Ourense, Spain Abstract Fifty-four honey samples collected over two consecutive harvest seasons ( ) from different floristic areas of Romania were analysed. A melissopalynological analysis including qualitative and quantitative analyses was carried out in order to identify the principal pollen types in Romanian honeys and therefore, the important plants exploited by Apis mellifera in this country. Seventy-seven pollen types from 35 botanical families were identified. The main pollen forms were: Brassica napus-type, Tilia, Helianthus annuus, Robinia pseudoacacia, Prunus, Castanea sativa, Fragaria-type and Plantago-type. Honey samples were classified as: acacia honey (Robinia pseudoacacia), lime honey (Tilia), rape honey (Brassica napus-type), sunflower honey (Helianthus annuus), cherry honey (Prunus). Three of them were honeydew honeys whereas the others were all polyfloral. The results from this study can be used as a palynological baseline data of Romanian artisanal honeys. Keywords: melissopalynological analysis, Romanian honeys, Robinia pseudoacacia, Tilia, Brassica napus In the period , Romania comes on the 4th position concerning honey export after Hungary (23.8 tons), Germany (23.7 tons) and Spain (13.8 tons). Romania also plays an important role regarding honey producers, contributing by tons to the EU honey production (Pîrvuţoiu & Popescu, 2011). In terms of apiculture, there are favourable conditions and melliferous resources in this country and in a normal year, the honey production reaches tons. Despite the importance of beekeeping in Romania, its honeys are poorly-studied. The most common commercial honeys are acacia, lime, rape, sunflower and fir honey. The three major vegetation zones in Romania are alpine, forest and steppe areas (Enescu, 2010). Forests cover about 27% of the territory. More than 69% are deciduous forests with oaks represented by different Quercus species (Q. robur, Q. petraea, Q. pedunculiflora, Q. cerris, Q. frainetto), Betula pendula, Fagus sylvatica, Larix, Carpinus or Fraxinus. Currently, Robinia pseudoacacia occupies ha and is located in the forest and plain areas being sporadic up to 400 m. Tilia occupies an area of ha and the most important culture of lime massive exists in Moldova (Mârză & Nicolaide, 1990). Some coniferous trees in mountain lands cover the 31% remaining of the forest. In these, Pinus sylvestris, Pinus cembra or Picea abies are the main species. Other well-represented taxa specific for the vegetation of the six Romanian provinces are Syringa josikaea, Artemisia santonica, Paeonia peregrina, Amygdalus nana, Chamaecytisus ratisbonensis, Ruscus aculeatus and Tamus communis. The Dobrogea province includes characteristic elements of the Danube Delta such as Phragmites australis or Typha latifolia. Other species in the area are Populus alba, Q. pedunculiflora, Fraxinus pallisae, Vitis sylvestris, Tilia tomentosa or Carpinus orientalis. A special feature of the province is represented by thorny bushes formed by Paliurus spina-christi, Berberis vulgaris and Crataegus monogyna among others. More than 60% of the land is used for agriculture. One third sustains permanent pastures and the rest is arable, more than half of which is planted with cereals, mainly maize and wheat. Around a tenth is covered with oilseeds, mainly Brassica napus Correspondence: Irina Dobre, Faculty of Food Science and Engineering, Applied Biotechnology and Biochemistry, Domneasca Street, 111 Building F, 101 Galati Romania , Romania. irina.dobre@ugal.ro (Received 19 January 2012; accepted 3 August 2012) 2013 Collegium Palynologicum Scandinavicum

3 114 I. Dobre et al. and Helianthus annuus. Other important crops are rice, oats, vineyard, sugar beet, melons, soy and cabbage. To help combat fraudulent labelling and to identify the geographical and botanical origin of honey, a method of melissopalynology was developed and proposed by the International Commission for Bee Botany (ICBB) (Louveaux et al., 1970). The pollen spectrum of a honey depends on the floral, agricultural and forest conditions where it is produced. The extent to which a given honey sample is derived from different plant sources can be deduced from the frequencies of the pollen and honeydew elements in it. Pollen identification has some limitations because it requires specialised and trained professional personnel with an extensive knowledge of pollen morphology (Cometto et al., 2003). In addition, the interpretation of pollen spectra can be difficult due to pollen grains of some plants being overrepresented, i.e. the percentage of pollen in the sediment is greater than the percentage of the corresponding nectar in honey. Examples of plants that are overrepresented include Castanea sativa, Eucalyptus, Brassica napus, while with some other species, the situation is reversed and they are underrepresented in the pollen spectrum. Species in this category include Robinia pseudoacacia, Tilia, Citrus or Rosmarinus (Louveaux et al., 1970; Ricciardelli d Albore & Vorwohl, 1979; Pérez-Arquillué et al., 1994; Persano-Oddo et al., 1995; von der Ohe et al., 2004). Palynological characterisation of honeys with different botanical origins (e.g. acacia, lime, chestnut, blackberry, eucalyptus, sunflower) has been deduced by many researchers from different countries (Maurizio, 1939, 1975; La Serna et al., 2002; Terrab et al., 2003; Atanassova & Kondova, 2004; Persano-Oddo & Piro, 2004; Downey et al., 2005; La Serna & Gómez-Ferreras, 2006; Seijo et al., 2011). However, in Romania little information concerning the melissopalynology is available in the literature. The purpose of this work is to contribute with new information to the palynological characterisation of Romanian honeys from different floristic areas, in order to determine the most important resources that are being exploited by Apis mellifera L. Material and methods Honey sampling The present study examined 54 different artisanal honey samples produced in Romania (in the regions of Moldova, Transilvania, Muntenia and Dobrogea) during These samples come from regions (Figure 1) with different types of vegetation and were taken directly from beekeepers or apicultural associations. The honey samples were aseptic transferred into plastic bottles and stored at 4 C until analyses. Melissopalynological analysis These analyses were carried out in the Laboratory of Aerobiology and Apiculture of the Faculty of Sciences, University of Vigo in Ourense, Spain. Both the quantitative and qualitative microscopical analyses were performed according to the method of Louveaux et al. (1970), using a non-acetolytic method with some adopted modifications concerning the time and the speed of centrifugation. Quantitative melissopalynological analysis Preparation of honey. Ten grams of honey with about 40 ml of distilled water were mixed, centrifuged at 4500 rpm (3383g) for 15 minutes, and the supernatant liquid was carefully removed. The residue was re-dissolved again and centrifuged for other 15 minutes. The preparation was made in duplicate for each sample. Quantitative analysis. Quantitative analysis was made using a volumetric method, with an aliquot of 10 µl for each residue per sample. The total quantity of pollen grains in the aliquots was counted using a light microscope at a magnification of 400. The average of aliquots is used to calculate the number of pollen grains per gram of honey (PG). Honeydew elements (HDE) consisting of fungal spores and algae were separately counted. Honey classification. Based on the total number of plant elements, honeys are placed into one of the following classes (Maurizio, 1939): class I with less than 2000 pollen grains per gram of honey (includes unifloral honeys with underrepresented pollen, e.g. Robinia and Citrus); class II with pollen grains including most of multifloral and honeydew honeys and mixtures of flower and honeydew honeys; class III with pollen grains includes unifloral honeys with overrepresented pollen and honeydew honeys; class IV with between including unifloral honey with strongly overrepresented pollen and some pressed honeys; and class V with more than pollen grains. Qualitative melissopalynological analysis This analysis was carried out using the entire residue obtained by centrifugation of 10 g of honey diluted

4 Honeys from Romania 115 Figure 1. The geographical regions of Romania. The asterisk indicates where the studied honeys originate from. in 40 ml of distilled water. The conditions were the same as for the quantitative analysis and light microscopy (400 or 1000, if necessary) was used to examine the samples. The pollen spectrum of each honey sample was determined by counting between 800 and 1000 pollen grains using the entire sediment. For all pollen species in the 54 samples, the individual occurrence was calculated and expressed as percentage. For each honey sample, the relative frequency classes were determined according to the international melissopalynological nomenclature using the terms: D dominant pollen (more than 45% of pollen grains counted), A accompanying pollen (representing 15 45% of the pollen spectrum), I important minor pollen (3 15%), R minor pollen (less than 3%) and pollen whose frequency is 1% or less should be quoted as present P. Statistical analysis All statistical analyses were performed with the STATGRAPHICS Centurion XVI software and SPSS Statistic 17.0 software for Windows. The most frequent pollen types were compared using an analysis of variance (ANOVA) and the Bonferroni test to determine the presence of statistically significant differences (p < 0.05) between samples. Principal component analysis (PCA) was used in order to study similarities between samples from the same botanical origin. Results Quantitatively, the total pollen content varied between 525 and pollen grains per gram of honey with an average of According to the Maurizio (1939) classification, 20 of 54 samples (37%) were poor in pollen and belonged to class I, 32 samples (59%) belonged to class II and only two samples (4%) fell into class III (Figure 2). This indicates that more than 50% of the studied honey samples had a medium pollen density, belonging to class II. There was a significant variation among the unifloral honeys: Acacia honey had minimum of 525 and maximum of 5475 pollen grains per gram of honey, lime honey had a minimum of 875 and maximum of 4975 pollen grains, while rape honey had a minimum of 700 and maximum of and Prunus had a pollen density of 5375 pollen grains per gram of honey. The honeydew honey was poor in pollen, recording an average of 2083 pollen grains, similar to lime honey, while the polyfloral honey had an average of 4044 pollen grains per gram of honey (Table I). The total pollen content of one of the rape samples recorded a maximum value ( pollen grains per gram), while the minimum value (525 pollen grains per gram) was found for acacia honey. The honeydew elements were counted separately. In this study, three samples seem to have similar characteristics to honeydew honeys. These were commercialised as fir, meadow and honeydew honeys. Fir honey had the higher HDE content (300 HDE/g). It should be mentioned that in these samples a major percentage of Quercus pollen was found. The most common fungal elements found were Cladosporium (found in almost all the samples), Aspergillus, Stemphylium, Alternaria, Pleospora and, in some of the samples, green algae such as Chlorococcus were also present.

5 116 I. Dobre et al. Qualitative pollen analysis revealed a total of 77 pollen types from 35 families. Families that occurred in more than 70% of samples include Brassicaceae (found in 100% of the honey samples), Polygonaceae (88.9%), Rosaceae (87%), Fabaceae (81.5%), Plantaginaceae (81.5%), Tiliaceae (77.8%) and Asteraceae (74.1%). Even though a wide variety of pollen types in different honey samples from different regions was encountered, only 21 taxa were the main sources for the bees food. These were present in more than 40% of samples. The best represented pollen types were Brassica napus-type, Rumex, Prunus, Rubus, Trifolium repens-type, Plantago, Fragaria-type, Robinia pseudoacacia, Tilia, Crataegus monogyna-type, Filipendulatype, Taraxacum officinale-type, Daucus carota-type, Helianthus annuus and Echium (Figure 3). The dominant pollen types were Brassica napus-type, Tilia, Helianthus annuus and Robinia pseudoacacia, which characterised the botanical origin of the studied samples (Table II). The presence of Prunus-type can be observed as an accompanying pollen type in six samples (9.3%); Quercus, Bifora radians, Castanea sativa and Echium in two samples, while Rubus, as important minor pollen, was present in 12 honey samples. Some pollen from non-nectariferous plants had low percentages in the pollen spectra of honeys. This is the case of Rumex, Plantago or Dactylis glomerata-type. The number of pollen forms per sample ranged from 12 (in one of the rape honeys) to 44 (in one lime honey) with an average value of 37. The botanical classification of honeys indicates that 12 would be classified as acacia honey (with Robinia pseudoacacia between 5% and 58% in the pollen spectrum), 14 as lime honey ( % of Tilia pollen), 11 as rape honey (with 52 93% of Brassica pollen), four as sunflower honey (with % of Helianthus annuus pollen), one as cherry honey (44.7% of Prunus pollen), three as honeydew honey and the rest as polyfloral honey (Table III). In the lime honeys, only two pollen types were present in all studied samples. These are Table I. Quantitative analysis of principal honey types. Type of honey Mean (PG/g honey) Minimum (PG/g honey) Maximum (PG/g honey) Maurizio s classes Lime I, II, III Acacia I, II Rape I, II, III Sun-flower I, II Honeydew I,II Polyfloral II Figure 2. Distribution of studied honeys into the Maurizio classificationofpollencontent. Brassica napus-type and Tilia. In all samples, Tilia occurred in percentages between 29 and 88.3%. The accompanying pollen in these samples was Brassica napus, Helianthus annuus and occasionally Anthriscus-type. Some other significant pollen types were Carduus-type, Bifora radians-type, Bellisperennis, Filipendula and Trifolium repens-type. In the acacia honey group, Robinia pseudoacacia, Brassica napus, Plantago, Rumex, Filipendula, Fragaria, Prunus and Rubus were present in all studied samples. Robinia pseudoacacia pollen was frequently secondary pollen with percentages ranging between 5 and 30%. In only one sample, the percentage of the pollen type exceeded 45% (58%). Brassica napus had high percentages of representation, frequently as accompanying or dominant pollen. It was also observed that some Rosaceae and some Fabaceae such as Trifolium or Vicia occurred in all the acacia honey samples. Brassica napus type was an overrepresented pollen with percentages ranging between 52 and 93% of the pollen spectra in rape honeys. Tilia was present in 9.1% of the rape samples as accompanying pollen. Prunus, Rubus, Helianthus annuus, Taraxacum officinale, Onobrychis viciifolia and Robinia pseudoacacia occurred in a number of samples with percentages exceeding 15%. For the sunflower honey group, Helianthus annuus, Taraxacum officinale-type, Chenopodium-type, Trifolium repens-type, Fragaria-type and Daucuscarota were present in 100% of the honey samples. Helianthus annuus was the dominant pollen with percentages ranging between 57.7 and 65.5%. Other

6 Honeys from Romania 117 Figure 3. Representation (%) of the principal pollen types identified in Romanian honey samples. important pollen types were Tilia, Brassica napus, Echium, Shymphytum officcinalis, Trifolium repens-type and Robinia pseudoacacia. Prunus, Brassica napus and Quercus were the principal pollen types in the cherry honey sample, and finally, some pollen types from mountain areas such as Castanea sativa and Quercus, Fabaceae and Trifolium repens-type, Brassica napus and Helianthus annuus were the main pollen types in honeydew honeys. Acacia, lime and sunflower honeys were produced in Muntenia, Transilvania and Moldova. Also one sample of lime was from Dobrogea. Rape honeys were common in all the studied regions. Honeydew honey was confined to Moldova and Transilvania, and the Cherry honey came from Transilvania. Discussion Pollen richness depends on the pollen production of the parent plant, the climatic conditions, the distance from the beehive to the flower field, the filtering by the bees proventriculus and, consequently, the diameter of the pollen grains and the mode of honey extraction (von der Ohe, 1994). If honey extraction takes place by pressing the combs, the pollen stored by the bees to feed the larvae, can pass in to the honey increasing significantly the pollen richness of the honey. In this case, pollen analysis is of little use in determining the botanical origin of product (Makhloufi et al., 2010). Pollen richness of the 54 studied samples revealed that the acacia, lime, sunflower and honeydew honey samples fell into class I and class II, rape honey samples belonged not only to the first two groups but also to class III, and the nine polyfloral samples belonged only to class II. In Argentina, Forcone et al. (2009) found multifloral honeys corresponding to these three groups. Persano-Oddo et al. (1995) reported that Robinia and Tilia have underrepresented pollen, so the percentages of the respective pollen in unifloral honeys are generally low to very low. It was observed in the present work that some acacia and lime honey samples also belonged to class II. This pollen richness was reported by Persano- Oddo and Piro (2004) and Was et al. (2011). Honeydew indicators were present in some samples and the most common fungi (Cladosporium, Alternaria and Stemphylium) have also been reported by many authors, who studied the fungal spores in detail (Méndez et al., 1997; Sabariego et al., 2004; Seijo et al., 2011). The HDE was between zero (for one lime and acacia honey, respectively) and 300 (for fir honey) with an average of 127. There are some exceptions, for example, one sample of acacia honey had a honeydew index (HDE/P) of 0.4 and the same value has been recorded elsewhere (Forcone et al., 2009). Only four pollen types were found as very frequent (Brassica napus-type, Tilia, Helianthus annuus, Robinia pseudoacacia) and seven taxa were classified as accompanying pollen (Prunus-type, Quercus, Castanea sativa, Echium, Trifolium repenstype, Filipendula-type, Vitis vinifera). Some pollen types of Asteraceae and Fabaceae families are highlighted having a notable representation in the examined samples, being the case of: Helianthus annuus, Carduus-type, Solidago, Bellis perennis, Taraxacum officinale-type, Centaurea cyanus, Robinia pseudoacacia, Trifolium repens-type and Vicia sativa-type. Some of these pollen types were also frequent in some Polish honeys (Wroblewska & Warakomska, 2009) and in honeys from Anatolia (Silici & Gökceoglu, 2007). The occurrence of Prunus and Filipendula-type in 87% of the samples

7 118 I. Dobre et al. Table II. Representation of the principal pollen types in the samples and their frequency classes. Family Pollen type P (<1) R (1 3) I (3 15) A (15 45) D > 45 Apiaceae Bifora radians Anthriscus-type Daucus carota-type Asteraceae Helianthus annuus Carduus-type Solidago Bellis perennis Taraxacum officinale-type Centaurea cyanus Boraginaceae Echium Symphytum officinalis-type Brassicaceae Brassica napus-type Sinapis Raphanus-type Fabaceae Robinia pseudoacacia Trifolium repens-type Onobrychis viciifolia Vicia sativa-type Melilotus Other Fabaceae Fagaceae Quercus Castanea sativa Lamiaceae Origanum-type Loranthaceae Loranthus Oleaceae Ligustrum Plantaginaceae Plantago Poaceae Dactylis glomerata-type Polygonaceae Rumex Fallopia Rhamnaceae Paliurus Rosaceae Prunus-type Filipendula Rubus Fragaria-type Crataegus monogyna-type Other Rosaceae Rubiaceae Galium-type Salicaceae Salix Tiliaceae Tilia Vitaceae Vitis vinifera with values higher than 1% revealed the importance of the Rosaceae in Romanian honeys. Most of the studied honeys were unifloral (78%). Monofloral honey samples were mostly from trees such as Robinia, Tilia, Prunus and from cultivated plants (Brassica napus, Helianthus annuus). Concerning the palynological characteristics of each honey type, it is worth mentioning the frequency of Rumex in Italian acacia honeys from the region Varese (Ricciardelli d Albore, 1988). Such large differences in the pollen content can be attributed to a number of factors: the amount of pollen present in the nectar can be very variable, pollen can be filtered out in the bee s honey sac (Maurizio, 1975) or the bee may take pollen without taking nectar (Anklam, 1998). In sunflower honeys, Helianthus annuus was present in all samples studied as dominant pollen and Tilia as secondary pollen. Tilia is a very common plant in Romania and its presence in Romanian honeys (not only in lime

8 Honeys from Romania 119 Figure 4. PCA of principal pollen types in the Romanian honeys. Projection into the plane composed by the principal axes: component 1 and component 2. Table III. Principal pollen types of monofloral honey and differences on pollen spectra. Honey type Number of samples Principal pollen type ANOVA (p < 0.05) Lime honey 14 Tilia Brassica napus Helianthus annuus Carduus-type Acacia honey 12 Brassica napus Robinia pseudoacacia Prunus Filipendula Castanea sativa Rape honey 11 Brassica napus Tilia Sun-flower honey 4 Helianthus annuus Tilia Brassica napus Cherry honey 1 Brassica napus Prunus Quercus Honeydew honey 3 Quercus Castanea sativa Trifolium repens-type Brassica napus Helianthus annuus Polyfloral honey 9 Brassica napus Bifora radians Tilia Prunus Plantago Echium 1, 3, 7 Brassica napus Robinia pseudoacacia 1 Helianthus anuus 4 1, 3, 4, 6, 7 Tilia Symphytum oficcinalis-type 4 Castanea sativa 6 Brassica napus 4 Robinia pseudoacacia Helianthus annuus 4 Tilia 2 2, 3, 4 1, 2, 4, 6, 7 Brassica napus Robinia pseudoacacia 1 Helianthus annuus 4 Tilia 2 Symphytum oficcinalis 4 Castanea sativa 6 Brassica napus 1,3, 7 Robinia pseudoacacia 1 1, 2, 3, 6, 7 Helianthus annuus Tilia 2 2, 3, 7 Symphytum oficcinalis Brassica napus 3 Robinia pseudoacacia 1 Helianthus annuus 4 Tilia 2 Castanea sativa 2, 3 Brassica napus 2,3, 4 Robinia pseudoacacia 1 Helianthus 4 Tilia 2 Prunus 2, 3 Superscript numbers show significance differences (p < 0.05) according to the Bonferroni test among the honey types. 1: acacia honey; 2: lime honey; 3: rape honey; 4: sun-flower honey; 5: cherry honey; 6: honeydew honey; 7: polyfloral honey.

9 120 I. Dobre et al. ones) in large amounts is to be expected. Lime honeys had values ranging from 28.3 to 88.3% of Tilia pollen. According to Persano-Oddo et al. (1995), honey from Tilia can even have no Tilia pollen at all, because of the sterility of many cultivated specimens of this plant. A similar situation is also found in Robinia percentages (from 5 to 57.9%). A PCA was performed with the most representative pollen types in the honeys studied (Figure 4). This procedure performs the extraction of four principal components with a total cumulative variance of 77.89%. This shows the projection of the principal pollen type into the plane composed by the first two principal components that explains 56.84% of the total variance. The distribution of the samples was performed by similarity in function of the pollen types, for which the ANOVA showed significant differences. The presence of this pollen in the pollen spectra of honeys leads to clearly differentiate the principal unifloral honeys. The ANOVA performed for the seven established groups revealed significant differences between the principal pollen types in honeys: Brassica napustype, Robinia pseudoacacia, Helianthus annuus and Tilia (Table III). The presence of Symphytum officinalis-type in sunflower honeys had also significant differences in lime, rape and polyfloral honeys. The same was revealed for Castanea sativa in honeydew honeys in relation to other honeys from mountain areas like lime honeys. A high variability among the samples was caused by the different nectar-pollenous and honeydew plants, which grow in the studied regions (Moldova, Muntenia, Transilvania and Dobrogea). For example, Quercus-type, which is a honeydew source, occurred in the three samples that were classified as honeydew honeys. These samples were collected from beekeepers in mountain areas of high altitude (Soria et al., 2004). There are no reports available concerning the palynological characteristics of Romanian honeys that make the present work being original in this field. It is noteworthy that the frequency of Brassica napus-type in all studied samples and the presence of Tilia and Robinia pseudoacacia has been also mentioned by other authors for Bulgarian (Atanassova & Kondova, 2004) and Croatian honeys (Sabo et al., 2011). Conclusion This work provides information on the pollen composition of Romanian honeys. The main honey producing plants are Brassica napus, Helianthus annuus, Prunus-type, Robinia pseudacacia and Tilia, which provides unifloral honeys. Honeys had low and very low pollen content in general. The presence of some taxa of Asteraceae, Fabaceae, Rosaceae, Lamiaceae and Apiaceae was also denoted; some of them are very common in Mediterranean areas. 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