Annals. Food Science and Technology 2017 MELLISOPALYNOLOGICAL ANALYSIS OF SOME COMMERCIAL HONEYS Abdullahi Alanamu Abdulrahaman*, Saheed Opeyemi Kolawole, Sheriff Babatunde Adeyemi, Hanifah Lola Misbahudeen Applied Plant Anatomy and Wood Technology Laboratory, Department of Plant Biology, Faculty of Life Sciences, University of Ilorin, Ilorin, Nigeria * E-mail: abdulrahamanaa@unilorin.edu.ng; Abstract Cases of adulterated honey sold in the markets is a crucial national concern as a source of food and health remedies. As part of efforts to monitor the level of adulteration in the neighbourhood, the present study is aimed at identifying the origin of the honey samples and ascertaining their authenticity. Laboratory and microscopic studies on 40 honey samples collected from 13 localities in Abeokuta (Majekodunmi, Itoku, Lafenwa, Somaarin, Odo-eran, Randa, Crescent University, Obantoko, Sanni, Iyana-cele, Osiele, Sabo and Atinsola), Ogun State and 15 places in Ibadan (University of Ibadan, Moor Plantation, Molete High School, Alegongo, Olegede, Agara, Ring Road, Aleshinloye, Basorun, Kasumu, Oremeji, Mapo, Apata, Oluyole, and Adelabu areas), Oyo State, South-West, Nigeria revealed that out of the 40 honey samples, 16 samples were adulterated with no pollen grains and the remaining 24 samples contained 12 pollen grain types from 81 plant taxa. In total, 168 pollen grains were extracted and identified. Some impurities were also observed in some of the honey samples. The frequency of the bee’s visit to each identified plant species was also determined based on the pollen grain frequency in each honey samples. The presence of the pollen grains in the 24 honey samples is a clear indication that those honeys are not adulterated but pure and multifloral. Key words: Adulterated honey, bee foraging, honey, pollen, mellisopalynology, pure honey Received: 07.05.2017 Received in revised form: 16.06.2017 1. INTRODUCTION Melissopalynology (i.e. the study of pollen in honey) is an important tool in determining the floral sources upon which the bees foraged to produce honey. Each flower species has a unique pollen grain, which using proper techniques, may be studied to determine the geographical origin and major floral sources of the honey. There are set procedures and laboratory guidelines for the study of pollen in honey and their relationship to the floral resources (Bryant and Jones, 2001). The study of pollen in a sample of honey makes it possible to gain evidence of the geographical location by observing the honey samples for the presence of varying combination of pollen that is typically only to that particular location (Louveaux et al., 1978). It is also possible to identify taxonomically the genera of the plants the honey bees visited, although honey may also contain airborne pollen from anemophilous plant species, spore and dust due to electrostatic charge of the Available on-line at www.afst.valahia.ro Accepted: 19.06.2017 worker bee. Information gained from a given honey sample is useful when substantiating claims of a particular honey source and is also of great importance for quality control and helps to ascertain whether honey is adulterated or not (Maurizio, 1951; Molan, 1998; Louveaux et al., 1978; Terrab et al., 2003). Honey samples may be mono-floral, bi-floral and hetero-floral or pluri-floral. Mono-floral honey may be more valuable than bi-floral honey and hetero-floral honeys (plurifloral) (Zet-Sche, 1932). There is no honey produced by bees flying free is entirely unifloral, unless the bees were reared in apiaries and exposed to a particular species of bee pollinated plant. In recent years, melisopalynology has attained a topic of global status. This is borne out of the fact that not only is honey useful as a food supplement; it is now increasingly being used in the treatment of various diseases (Molan, 2001). These healing properties of honey are as a result of the integration of pollen and nectar containing bio active ingredients from medicinal Honey is a natural sweet substance 493 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 and is produced by honeybees from the nectar of blossoms, from secretion of living parts of plants. Although honey contains many chemical components such as carbohydrates i.e. glucose, maltose, sucrose, fructose and other complex carbohydrates, vitamins and minerals, the specific composition of any batch of honey depends on the flowers available to the bees that produced the honey. In many cultures, honey has associations that go beyond its use as a food. Honey is frequently a talisman and symbol of sweetness. In Nigeria, the 3 most common ethnic groups (Yoruba, Igbo and Hausa) use honey during occasions like naming ceremony of new born baby, as one of the requirements in paying bride price during a marriage ceremony, honey is also used as a gift. For at least 2700 years, honey has been used by humans to treat a variety of ailments through topical application, but only recently has the antiseptic and antibacterial properties of honey chemically explained (Jusbin, 1996; Abdulla and AbdulAziz, 1998; Wahdan, 1998). This present study is a further contribution to the melissopalynological investigations of honey samples in Nigeria, focusing on the honey samples collected from 40 localities in Ibadan and Abeokuta, Nigeria. Therefore, the study is aimed at identifying the origin of the honey samples and ascertaining their authenticity. 2. MATERIALS AND METHODS Forty honey samples were collected from twenty areas, each in Ibadan, Oyo State and Abeokuta, Ogun State, South-West, Nigeria. The 15 samples from Ibadan include those collected from University of Ibadan, Moor Plantation, Molete High School, Alegongo, Olegede, Agara, Ring Road, Aleshinloye, Basorun, Kasumu, Oremeji, Mapo, Apata, Oluyole, and Adelabu areas, and 13 samples from Abeokuta included honeys from Majekodunmi, Itoku, Lafenwa, Somaarin, Odoeran, Randa, Crescent University, Obantoko, Sanni, Iyana-cele, Osiele, Sabo and Atinsola. Available on-line at www.afst.valahia.ro Melissopalynological studies were performed on the 28 samples. Distilled water was warmed on a Bunsen burner. Five grams (5g) of each honey sample was weighed using the weighing balance into a beaker. 10mls of the hot distilled water was added and stirred by manual shaking till the honey samples dissolved totally. The solution was poured into the centrifuge tube and placed in the centrifuge. It was allowed to spin at 3000rpm for 5 minutes. The supernatant fluid was discarded. To the residue, 3-5ml glacial Acetic acid was added, stirred and allowed to stand for 5 minutes. The solution was centrifuged again, with the supernatant fluid being discarded. Acetolysis mixture was added to the residue in a fume cupboard, boiled in the water bath at 80-900c for 2-3 minutes. The mixture was centrifuged and decanted. To the residue, distilled water was added thrice to rinse the residue, centrifuging and decanting at each time. The final residue was mounted on a slide with the aid of dropper or micropipette and allowed to dry. Few drops of Isopropyl alcohol were added to the dried residue, and allowed to dry. Then a few drops of already warmed liquid glycerine were added to the slide as a mounting agent for observation (Erdtman, 1960). The slide was covered using a coverslip. The slide was made permanent by using nail varnish as sealant on the edges of the coverslip. The slide was taken for microscopic examination, identification, and frequency of the pollen species. Micrographs were taken using the Amscope camera and pollen images were cropped out for identification. The slides were labeled accordingly. Acetolysis mixture: this was prepared by addition of concentrated sulphuric acid to acetic anhydride in the ratio 9:1. Liquid glycerin: This was prepared by the addition of distilled water to liquid glycerol. Pollen observed in the honey samples were identified using some pollen atlas such as Dieter et al., 2013; and William et al., 2013. Using 35 snaps of field observation at 40x magnification as quadrat, the number of pores on pollens was noted to determine the frequency of the different pollen types present. 494 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 The frequency of each pollen type was expressed as the percentage occurrence of such pollen type based on all occurrences. For the presentation of frequencies of pollen grains in honey, the system adopted by Louveaux et al. (1978) was used. The percentage frequency of each pollen type was determined using this formula: remaining 24 samples analyzed in the study were found to contain pollen grains of 81 plant taxa and 12 pollen types. The identified taxa belong to varying genera of native herbs, shrubs, grass and trees. These pollens are of varying shapes, sizes and morphological features, suggesting that the honey samples are multifloral (Tables 1-24; Plates 1-24). %Frequency = Number of pollen of a taxon x 100 Total number of all pollens Apata honey Five pollen types of eight plant species belonging to seven families were observed in the honey samples collected from Apata area. Pollen grains from Gerradima foliosa is more dominant (28.3%) followed by pollen grains from Mangifera indica (18.9%) and pollen grains from Amaranthus spp. being the least with 0.3% frequency (Table 1; Plate 1). The following terms have been used in the estimation of PK (pollen grain) frequencies: “very frequent” for grains constituting more than 45% of the total, “sporadic or rare for grain constituting less than 3% and the following terms used for frequency classes. The term „‟predominant pollen” (more than 45% of the pollen grains counted), ‟secondary or accompanying pollen‟‟ (16-45%), ‟important minor or important isolated pollen’’ (3-15%), ‟minor or isolated pollen’’ (less than 3%) (Vergeron, 1964). Necessary precautions were taken to avoid contamination of one pollen source from other pollen sources. Kasumu honey Six pollen types from eight plant species belonging to six families were observed in the honey samples collected from Kasumu area. Pollen grains from Tilia americana is more dominant (26.8%) followed by pollen grains from Combretum guienzii (17.1%) and pollen grains from Acidanthera brevicollis being the least with 4.2% frequency (Table 2; Plate 2). 3. RESULTS AND DISCUSSION 3.1. RESULTS Of the 40 honey samples collected, 16 samples were adulterated with no pollen grains and the a b f c g d h e i Plate 1: Pollen samples (a- Amaranthus sp, b- Zea mays, c- Gerradina foliosa d- Mangifera indica, e- Tetrochidium didymostemon, g- Chaemacyparis nootkatensis, h- Lannea acida, i- Monotes kerstingii) and impurity (f- leaf-like) obtained from the honey collected in Apata, Ibadan, Nigeria. Available on-line at www.afst.valahia.ro 495 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b c d g h e f i Plate 2: Pollen samples (a- Combretum guienzii, c- Trema guineensis, d- Gunnera chilensis, e- Acidanthera brevicollis, f- Entada abyssinica, g- Tilia Americana, h- Parkia inundabilis, i- Piptadenia Africana) and impurity (b- a leaf-like), obtained from the honey collected in Kasumu, Ibadan, Nigeria. Table 1: Plant sources, types and frequency of pollens obtained from Apata honey Plant species Amaranthus spp. Zea mays Gerradina foliosa Mangifera indica Tetrochidium didymostemon Chaemacyparis nootkatensis Lannea acida Monotes kerstingii Family Amaranthaceae Poaceae Gerrardinaceae Anacardiaceae Euphorbiaceae Cupressaceae Anacardiaceae Dipterocarpaceae Pollen type Pantoporate Monoporate Tricolporate Tricolporate Tricolporate Monocolpate Tricolporate Tricolpate Frequency (%) 0.3 14.6 28.3 18.9 15.6 7.3 9.2 5.8 Table 2: Plant sources, types and frequency of pollens obtained from Kasumu honey Plant species Combretum guienzii Trema guineensis Gunnera chilensis Acidanthera brevicollis Entada abyssinica Tilia americana Parkia inundabilis Piptadenia africana Family Combretaceae Cannabaceae Gunneraceae Iridaceae Fabaceae Tiliaceae Fabaceae Fabaceae Lafenwa honey Three pollen types of five plant species belonging to three families were observed in the honey samples collected from Lafenwa area. Pollen grains from Acidanthera brevicollis is more dominant (35.4%) followed by pollen grains from Piptadenia africana (21.0%) and pollen grains from Clifftortia nitidula being the least with 9.8% frequency (Table 3; Plate 3). Molete High School honey Two pollen types of six plant species belonging to five families were observed in the honey Available on-line at www.afst.valahia.ro Pollen type Heterocolporate Diporate Tricolpate Monocolpate Triporate Tricolpate Tricolporate Tricolporate Frequency (%) 17.1 15.8 13.1 4.2 6.6 26.8 7.3 9.1 samples collected from Molete high school area. Pollen grains from Typha capensis is more dominant (25.7%) followed by pollen grains from Typha augustifolia (21.2%) and pollen grains from Gerradima foliosa being the least with 3.5% frequency (Table 4; Plate 4). Moor plantation honey Eight pollen types of nine plant species belonging to seven families were observed in the honey samples collected from Moor Plantation area. Pollen grains from Clausena anisata is more dominant (21.8%) followed by pollen grains from Rhizophora mangle (15.3%) 496 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 and pollen grains from Clifftortia nitidula being the least with 3.5% frequency (Table 5; Plate 5). grains from Gunnera chilenis being the least with 3.6% frequency (Table 6; Plate 6). Oremeji honey Four pollen types of six plant species belonging to six families were observed in the honey samples collected from Oremeji area. Pollen grains from Acidanthera brevicollis is more dominant (29.5%) followed by pollen grains from Lannea acida (21.2%) and pollen grains from Elaeis guineensis being the least with 3.6% frequency (Table 7; Plate 7). Ologede honey Five pollen types of eight plant species belonging to eight families were observed in the honey samples collected from Ologede area. Pollen grains from Berlinia bifoliata is more dominant (24.1%) followed by pollen grains from Hymenocardia acida (19.3%) and pollen a b c d e Plate 3: Pollen samples (a- Acidanthera brevicollis, b- Piptadenia africana c- Clifftortia nitidula d- Poliostigma reticulatum e- Parkia bussei) obtained from the honey collected in Lafenwa, Abeokuta, Nigeria a b c d e f g Plate 4: Pollen samples (a- Typha capensis, b- Lannea acida, d-Typha augustifolia, e- Gerradina foliosa, f- Grewia glandulosa, g- Piptadenia africana) and impurity (c- leaf-like) obtained from the honey collected in Molete High School, Ibadan, Nigeria a b c d e f g h i Plate 5: Pollen samples (a- Hymenocardia acida, b- Parkia bussei, c- Clifftortia nitidula, d- Jatropha curcas, eCombretum spp, f- Rumex acetosella, g- Clausena anisata, h- Rhizophora mangle, i- Terminalia aemula) obtained from the honey collected in Moor plantation, Ibadan, Nigeria. Available on-line at www.afst.valahia.ro 497 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b c d e f g h i Plate 6: Pollen samples (b- Lannea acida, c- Hymenocardia acida, d- Gunnera chilensis, e- Berlinia bifoliolata, fSyzygium guineense, g- Typha capensis, h- Aesculus carnifornica, i- Securidaca longependuculata) and impurity (a- leaf-like) obtained from the honey collected in Ologede, Ibadan, Nigeria. a b d d e f Plate 7: Pollen samples (a- Acidanthera brevicollis, b- Elaeis guineensis, c- Piptadenia Africana, d- Hymenocardia acida, e- Lannea acida, f- Typha capensis) obtained from the honey collected in Oremeji, Ibadan, Nigeria. Table 3: Plant sources, types and frequency of pollens obtained from Lafenwa honey Plant species Acidanthera brevicollis Piptadenia africana Clifftortia nitidula Poliostigma reticulatum Parkia bussei Family Iridaceae Fabaceae Rosaceae Fabaceae Fabaceae Pollen type Monocolpate Tricolporate Tricolporate Triporate Tricolporate Frequency (%) 35.4 21.0 9.8 15.6 18.2 Table 4: Plant sources, types and frequency of pollens obtained from Molete High school honey Plant species Typha capensis Lannea acida Typha augustifolia Gerradina foliosa Grewia glandulosa Piptadenia africana Family Typhaceae Anacardiaceae Typhaceae Gerrardinaceae Malvaceae Fabaceae Pollen type Monoporate Tricolporate Monoporate Tricolporate Tricolporate Tricolporate Frequency (%) 25.7 13.8 21.2 3.5 15.9 19.9 Table 5: Plant sources, types and frequency of pollens obtained from Moor Plantation honey Plant species Hymenocardia acida Parkia bussei Clifftortia nitidula Jatropha curcas Combretum spp. Rumex acetosella Clausena anisata Rhizophora mangle Terminalia aemula Family Euphorbiaceae Fabaceae Rosaceae Euphorbiaceae Combretaceae Polygonaceae Rutaceae Rhizophoraceae Combretaceae Available on-line at www.afst.valahia.ro Pollen type Triporate Heterocolpate Tricolporate Panporate Heterocolporate Paniculate Tricolporate Diporate Monocolpate 498 Frequency (%) 7.3 12.1 4.3 11.8 5.9 15.3 21.8 15.3 6.2 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Table 6: Plant sources, types and frequency of pollens obtained from Ologede honey Plant species Lannea acida Hymenocardia acida Gunnera chilensis Berlinia bifoliolata Syzygium guineense Typha capensis Aesculus carnifornica Securidaca longependuculata Family Anacardiaceae Phyllanthaceae Gunneraceae Fabaceae Myrtaceae Typhaceae Sapindaceae Polygalaceae Pollen type Tricolporate Triporate Tricolpate Tricolporate Tricolpate Monoporate Tricolporate Stephanocolporate Frequency (%) 9.5 19.3 3.6 24.1 12.3 11.4 15.6 4.2 Table 7: Plant sources, types and frequency of pollens obtained from Oremeji honey Plant species Acidanthera brevicollis Elaeis guineensis Piptadenia africana Hymenocardia acida Lannea acida Typha capensis Family Iridaceae Arecaceae Fabaceae Phyllanthaceae Anacardiaceae Typhaceae University of Ibadan honey Four pollen types of seven plant species belonging to six families were observed in the honey samples collected from University of Ibadan area. Pollen grains from Turraeanthus africana is more dominant (26.7%) followed by pollen grains from Typha capensis (21.2%) and pollen grains from Passiflora spp. being the least with 2.7% frequency (Table 8; Plate 8). Alegongo honey Four pollen types of seven plant species belonging to five families were observed in the honey samples collected from Alegongo area. Pollen grains from Elaeis guineensis is more dominant (24.5%) followed by pollen grains from Milletia oblata (18.9%), and pollen grains from Ginkgo biloba being the least with 7.4% frequency (Table 9; Plate 9). Atinsola honey Three pollen types of seven plant species belonging to six families were observed in the honey samples collected from Atinsola area. Pollen grains from Alchornea cordifolia is more dominant (28.2%) followed by pollen grains from Hymenocardia acida (23.6%) and pollen grains from Adenia nicobarrica being Available on-line at www.afst.valahia.ro Pollen type Monocolpate Monocolpate Tricolporate Triporate Tricolporate Monoporate Frequency (%) 29.5 8.3 11.6 19.8 21.2 9.6 the least with 2.4% frequency (Table 10; Plate 10). Crescent University honey Seven pollen types of twelve plant species belonging to twelve families were observed in the honey samples collected from Crescent University area. Pollen grains from Caryolus cornuta is more dominant (15.6%) followed by pollen grains from Protea sussane (13.7%) and pollen grains from Acridocarpus macrocalyx being the least with 1.2% frequency (Table 11; Plate 11). Itoku honey Three pollen types of five plant species belonging to four families were observed in the honey samples collected from Itoku area. Pollen grains from Piptadenia africana is more dominant (38.8%) followed by pollen grains from Typha augustifolia (19.3%) and pollen grains from Entada abyssinica being the least with 7.9% frequency (Table 12; Plate 12). Iyana cele honey Four pollen types of five plant species belonging to five families were observed in the honey samples collected from Iyana-cele area. Pollen grains from Acridocarpus macrocalyx is 499 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 pollen grains from Xylla evansii is the least with 3.8% frequency (Table 14; Plate 14). Basorun honey Four pollen types of six plant species belonging to six families were observed in the honey samples collected from Basorun area. Pollen grains from Cunnonia pterophylla is more dominant (33.3%) followed by pollen grains from Guiera senegalensis (19.8%) with pollen grains from Sterculia tragacantha being the least with 6.9% frequency (Table 15; Plate 15). more dominant (33.3%) followed by pollen grains from Vitex doniana (23.8%) and pollen grains from Parkia velutina being the least with 5.3% frequency (Table 13; Plate 13). Majekodunmi honey One pollen type of six plant species belonging to three families was observed in the honey samples collected from Majekodunmi area. Pollen grains from Prosopis africana is more dominant (39.3%) followed by pollen grains from Zanthoxylum procerum (18.8%) and Table 8: Plant sources, types and frequency of pollens obtained from University of Ibadan honey Plant species Passiflora spp. Typha augustifolia Typha capensis Amaranthus spp. Turraeanthus africana Prunus africana Vitex ambionensis Family Passifloraceae Typhaceae Typhaceae Amaranthaceae Meliaceae Rosaceae Lamiaceae Pollen type Heterocolpate Monoporate Monoporate Pantoporate Triporate Tricolporate Tricolpate Frequency (%) 2.7 15.9 21.2 7.5 26.7 17.1 8.9 Table 9: Plant sources, types and frequency of pollens obtained from Alegongo honey Plant species Piliostigma reticulatum Rhynchosia spp. Ginkgo biloba Elaeis guineensis Elaeodendron buchananii Protea susannae Milletia oblata Family Fabaceae Fabaceae Ginkgoaceae Arecaceae Celestraceae Proteaceae Fabaceae a b e f Pollen type Triporate Tricolporate Dicolporate Monocolpate Tricolporate Triporate Tricolporate c g Frequency (%) 12.3 13.8 7.4 24.5 14.2 8.9 18.9 d h Plate 8: Pollen samples (a- Passiflora spp., b- Typha augustifolia, c- Typha capensis, d-Amaranthus spp., eTurraeanthus africana, f- Prunus africana, h-Vitex ambionensis) and impurity (g-leaf-like) obtained from the honey collected in University of Ibadan, Ibadan, Nigeria. Available on-line at www.afst.valahia.ro 500 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b c e d f g Plate 9: Pollen samples (a-Piliostigma reticulatum, b-Rhynchosia spp., c-Ginkgo biloba, d-Elaeis guineensis, eElaeodendron buchananii, f-Protea susannae, g-Milletia oblata) obtained from the honey collected in Alegongo, Ibadan, Nigeria. Table 10: Plant sources, types and frequency of pollens obtained from Atinsola honey Plant species Adenia nicobarrica Parkia inundabillis Actinidia palmatum Alchornea cordifolia Entada umbonata Acridocarpus macrocalyx Hymenocardia acida Family Passifloraceae Fabaceae Actinidiaceae Euphorbiaceae Fabaceae Malpighiaceae Phyllanthaceae Pollen type Tricolporate Tricolpate Tricolporate Tricolpate Triporate Tricolporate Triporate Frequency (%) 2.4 7.3 15.7 28.2 11.3 11.5 23.6 Table 11: Plant sources, types and frequency of pollens obtained from Crescent University honey Plant species Huperzia spp. Parinari curatellifolia Jatropha curcas Typha augustifolia Elaeis guineensis Premna maxima Acidocarpus macrocalyx Alcea rosea Hippocratea affinis Agelaea heterophylla Caryolus cornuta Protea susannae Family Huperziaceae Chrysobalanaceae Euphorbiaceae Typhaceae Arecaceae Lamiaceae Malpighiaceae Malvaceae Celastraceae Connaraceae Betulaceae Proteaceae Pollen type Tricolpate Tricolporate Panporate Monoporate Monocolpate Tricolpate Tricolporate Monocolpate Tricolporate Tricolporate Tricolpate Triporate Frequency (%) 2.2 7.6 10.8 5.3 8.5 4.2 1.2 3.0 11.9 6.2 15.6 13.7 Table 12: Plant sources, types and frequency of pollens obtained from Itoku honey Plant species Entada abyssinica Gerradina foliosa Piptadenia africana Hymenocardia acida Typha augustifolia Family Fabaceae Gerrardinaceae Fabaceae Phyllanthaceae Typhaceae Available on-line at www.afst.valahia.ro 501 Pollen type Tricolporate Tricolporate Tricolporate Triporate Monoporate Frequency (%) 7.9 17.3 38.8 16.7 19.3 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Table 13: Plant sources, types and frequency of pollens obtained from Iyana cele honey Plant species Vitex doniana Celtis integrifolia Parkia velutina Acridocarpus macrocalyx Jatropha curcas Family Lamiaceae Cannabaceae Fabaceae Malpighiaceae Euphorbiaceae Pollen type Tricolpate Monoporate Tricolpate Tricolporate Panporate Frequency (%) 23.8 19.2 5.3 33.3 18.4 Table 14: Plant sources, types and frequency of pollens obtained from Majekodunmi honey Plant species Xylla evansii Entada abyssinica Zanthoxylum procerum Prosopis africana Hippocratea affinis Rhynchosia spp. Family Fabaceae Fabaceae Rutaceae Fabaceae Celastraceae Fabaceae Pollen type Tricolporate Tricolporate Tricolporate Tricolporate Tricolporate Tricolporate Frequency (%) 3.8 9.6 18.8 39.3 17.2 11.3 Table 15: Plant sources, types and frequency of pollens obtained from Basorun honey Plant species Sterculia tragacantha Typha augustifolia Mangifera indica Cunonia pterophylla Guiera senegalensis Rhizophora mangle Family Malvaceae Typhaceae Anacardiaceae Cunoniaceae Combretaceae Rhizophoraceae a Pollen type Tricolporate Monoporate Tricolporate Monoporate Heterocolporate Diporate b Frequency (%) 6.9 11.8 16.7 33.3 19.8 11.5 c e f d g Plate 10: Pollen samples (a-Adenia nicobarrica, b-Parkia inundabillis, c-Actinidia palmatum, d-Alchornea cordifolia, e-Entada umbonata, f-Acridocarpus macrocalyx, g-Hymenocardia acida) obtained from the honey collected in Atinsola, Abeokuta, Nigeria. Available on-line at www.afst.valahia.ro 502 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b g c h d i e j f k l Plate 11: Pollen samples (a-Huperzia spp., b-Parinari curatellifolia, c-Jatropha curcas, d-Typha augustifolia, eElaeis guineensis, f-Premna maxima, g-Acidocarpus macrocalyx, h-Alcea rosea, i-Hippocratea affinis, j-Agelaea heterophylla, k-Caryolus cornuta, l-Protea susannae) obtaiined from the honey collected in Crescent University, Abeokuta, Nigeria. a b c d e Plate 12: Pollen samples (a-Entada abyssinica, b-Gerradina foliosa, c-Piptadenia africana, d-Hymenocardia acida, e-Typha augustifolia) obtained from the honey collected in Itoku, Abeokuta, Nigeria. a b c d e Plate 13: Pollen samples (a-Vitex doniana, b-Celtis integrifolia, c-Parkia velutina, d-Acridocarpus macrocalyx, eJatropha curcas) obtained from the honey collected in Iyana cele, Abeokuta, Nigeria. a b c d e f Plate 14: Pollen samples (a-Xylla evansii, b-Entada abyssinica, c-Zanthoxylum procerum, d-Prosopis africana, eHippocratea affinis, f-Rhynchosia spp.) obtained from the honey collected in Majekodunmi, Abeokuta, Nigeria. Available on-line at www.afst.valahia.ro 503 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b c d e f g Plate 15: Pollen samples (a-Sterculia tragacantha, b-Typha augustifolia, c-Mangifera indica, d-Cunonia pterophylla, f-Guiera senegalensis, g-Rhizophora mangle) and impurity (e- leaf-like) obtained from the honey collected in Basorun, Ibadan, Nigeria. a b c d e f Plate 16: Pollen samples (a-Phoenix reclinata, b-Indigofera leptoclada, c-Typha augustifolia, d-Scytopetalum tieghemii, e-Alchornea chordifolia, f-Aesculus indica) obtained from the honey collected in Obantoko, Abeokuta, Nigeria. a b c d e Plate 17: Pollen samples (a-Parkia velutina, b-Terminalia brownii, c-Agrimonia eupatoria, d-Terminalia aemula, ePiptadenia africana) obtained from the honey collected in Oluyole, Ibadan, Nigeria. a b c e f d g Plate 18: Pollen samples (a-Combretum aculeatum, b-Vitex doniana, c-Gomphrena celosioides, d-Raphia ruffia, eAcridocarpus macrocalyx, f-Lotus arabicus, g-Grewia glandulosa) obtained from the honey collected in Osiele, Abeokuta, Nigeria. Available on-line at www.afst.valahia.ro 504 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Table 16: Plant sources, types and frequency of pollens obtained from Obantoko honey Plant species Phoenix reclinata Indigofera leptoclada Typha augustifolia Scytopetalum tieghemii Alchornea chordifolia Aesculus indica Family Arecaceae Fabaceae Typhaceae Lecythidaceae Euphorbiaceae Sapindaceae Pollen type Monocolpate Tricolporate Monoparate Tricolporate Tricolporate Monocolpate Frequency (%) 16.7 23.6 8.3 11.3 29.2 10.9 Table 17: Plant sources, types and frequency of pollens obtained from Oluyole honey Plant species Parkia velutina Terminalia brownii Agrimonia eupatoria Terminalia aemula Piptadenia africana Family Fabaceae Combreataceae Rosaceae Combretaceae Fabaceae Pollen type Tricolpate Heterocolporate Tricolporate Monocolpate Tricolporate Frequency (%) 6.9 12.7 25.8 13.4 41.2 Table 18: Plant sources, types and frequency of pollens obtained from Osiele honey Plant species Combretum aculeatum Vitex doniana Gomphrena celosioides Raphia ruffia Acridocarpus macrocalyx Lotus arabicus Grewia glandulosa Family Combretaceae Lamiaceae Amaranthaceae Aracaceae Malpighiaceae Fabaceae Malvaceae Obantoko honey Three pollen types of six plant species belonging to six families were observed in the honey samples collected from Obantoko area. Pollen grains from Alchornea cordifolia is more dominant (29.2%) followed by pollen grains from Indigofera leptoclada (23.6%) with pollen grains from Typha augustifolia being the least with 8.3% frequency (Table 16; Plate 16). Oluyole honey Four pollen types of five plant species belonging to three families were observed in the honey samples collected from Oluyole area. Pollen grains from Piptadenia africana is more dominant (41.2%) followed by pollen grains from Agrimonia eupatoria (25.8%) with pollen grains from Parkia velutina being the least with 6.9% frequency (Table 17; Plate 17) Available on-line at www.afst.valahia.ro Pollen type Heterocolparate Tricolporate Pantoporate Monocolpate Tricolporate Tricolporate Tricolporate Frequency (%) 5.2 13.3 16.4 7.3 9.5 11.1 37.2 Osiele honey Four pollen types of seven plant species belonging to seven families were observed in the honey samples collected from Osiele area. Pollen grains from Grewia glandulosa is more dominant (37.2%) followed by pollen grains from Gomphrena celosioides (16.4%) with pollen grains from Combretum aculeatum being the least with 5.2% frequency (Table 18; Plate 18). Randa honey Five pollen types of nine plant species belonging to nine families were observed in the honey samples collected from Randa area. Pollen grains from Entada abyssinica is more dominant (24.0%) followed by pollen grains from Celtis integrifolia (18.3%) with pollen grains from Vitex doniana being the least with 5.4% frequency (Table 19; Plate 19). 505 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Somaarin honey Four pollen types of seven plant species belonging to six families were observed in the honey samples collected from Somaarin area. Pollen grains from Typha augustifolia is more dominant (23.6%) followed by pollen grains from Typha capensis (18.1%) with pollen grains from Pteleopsis diptera being the least with 8.3% frequency (Table 20; Plate 20). Odo-eran honey Five pollen types of seven plant species belonging to seven families were observed in the honey samples collected from Odo-eran area. Pollen grains from Typha augustifolia is more dominant (26.3%) followed by pollen grains from Acidanthera brevicollis (20.7%) with pollen grains from Lannea acida being the least with 4.9% frequency (Table 21; Plate 21). Sanni honey Five pollen types of seven plant species belonging to six families were observed in the honey samples collected from Sanni area. Pollen grains from Acridocarpus macrocalyx is more dominant (24.6%) followed by pollen a grains from Entada abyssinica (19.8%) with pollen grains from Phaeoceros laevis being the least with 3.2% frequency (Table 22; Plate 22). Aleshinloye honey Six pollen types of six plant species belonging to six families were observed in the honey samples collected from Aleshinloye area. Pollen grains from Trema guineensis is more dominant (27.6%) followed by pollen grains from Parkia inundabilis (23.9%) with pollen grains from Tylophora sylvatica being the least with 7.4% frequency (Table 23; Plate 23). Agara honey Seven pollen types of nine plant species belonging to nine families were observed in the honey samples collected from Agara area. Pollen grains from Ipomoea orchracea is more dominant (17.1%) followed closely by pollen grains from Justica cordata (15.4%) with pollen grains from Combretum guienzi being the least with 3.5% frequency (Table 24; Plate 24). b c d e f g h i Plate 19: Pollen samples (a- Celtis integrifolia, b-Xyris Montana, c-Entada abyssinica, d-Syzigium guineense, ePhaeceros laevis, f-Erica arborea, g-Vitex doniana, h-Gerradina foliosa, i-Acidanthera brevicollis) obtained from the honey collected in Randa, Abeokuta, Nigeria. a b c d e f g Plate 20: Pollen samples (a-Acidocarpus macrocalyx, b-Typha augustifolia, c-Pteleopsis diptera, d-Typha capensis, e-Lannea acida, f- Indigofera leptoclada, g-Xyris Montana) bgobtained from the honey collected in Somaarin, Abeokuta, Nigeria Available on-line at www.afst.valahia.ro 506 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 a b c d e f g Plate 21: Pollen samples (a-Typha augustifolia, b-Terminalia aemula, c-Acacia eglengii, d-Lannea acida, eAcidanthera brevicollis, f-Elaeis guineensis, g-Celtis integrifolia) obtained from the honey sample collected in Odo eran, Abeokuta, Nigeria a b c d e f g Plate 22: pollen samples (a-Acridocarpus macrocalyx, b-Entada abyssinica, c-Polygonum lapathifolum, dAnthosthema senegalense, e-Xyris Montana, f-Phaeoceros laevis, g- Parkia inundabilis) obtained from the honey collected in Sanni, Abeokuta, Nigeria. a b c d e f Plate 23: Pollen samples (a-Parkia inundabilis, b-Acidanthera brevicollis, c-Terminalia brownii, d-Gerradina foliosa, e-Tylophora sylvatica, f-Trema guineesis) obtained from the honey collected in Aleshinloye, Ibadan, Nigeria a b c d e f g h i Plate 24: Pollen samples (a-Tilia americana, b-Erica arborea, c-Celtis integrifolia, d-Elaeis guineensis, eCombretum gueinzii, f-Ipomoea ochracea, g-Gomphrena celosioides, h-Carya sp, i-Justica cordata) obtained from the honey collected in Agara, ibadan, Nigeria. Available on-line at www.afst.valahia.ro 507 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Table 19: Plant sources, types and frequency of pollens obtained from Randa honey Plant species Celtis integrifolia Xyris Montana Entada abyssinica Syzigium guineense Phaeceros laevis Erica arborea Vitex doniana Gerradina foliosa Acidanthera brevicollis Family Cannabaceae Xyridaceae Fabaceae Myrtaceae Notothyladaceae Ericaceae Lamiaceae Gerrardinaceae Iridacaeae Pollen type Triporate Monocolpate Tricolporate Tricolpate Inaperturate Tricolpate Tricolpate Tricolporate Monocolpate Frequency (%) 18.3 10.8 24.0 6.7 8.5 6.3 5.4 12.8 7.2 Table 20: Plant sources, types and frequency of pollens obtained from Somaarin honey Plant species Acidocarpus macrocalyx Typha augustifolia Pteleopsis diptera Typha capensis Lannea acida Indigofera leptoclada Xyris Montana Family Malpighiaceae Typhaceae Combretaceae Typhaceae Anacardiaceae Fabaceae Xyridaceae Pollen type Tricolporate Monoporate Heterocolporate Monoporate Tricolporate Tricolporate Monocolpate Frequency (%) 13.8 23.6 8.3 18.1 17.3 8.7 10.2 Table 21: Plant sources, types and frequency of pollens obtained from Odo eran honey Plant species Typha augustifolia Terminalia aemula Acacia eglengii Lannea acida Acidanthera brevicollis Elaeis guineensis Celtis integrifolia Family Typhacae Combretaceae Fabaceae Anacardiaceae Iridaceae Arecaceae Cannabaceae Pollen type Monoporate Heterocolporate Tricolporate Tricolporate Monocolpate Monocolpate Triporate Frequency (%) 26.3 13.4 6.7 4.9 20.7 19.8 8.2 Table 22: Plant sources, types and frequency of pollens obtained from Sanni honey Plant species Acridocarpus macrocalyx Entada abyssinica Polygonum lapathifolum Anthosthema senegalense Xyris Montana Phaeoceros laevis Parkia inundabilis Family Malpighiaceae Fabaceae Polygonaceae Euphorbiaceae Xyridaceae Notothyladaceae Fabaceae Pollen type Tricolporate Tricolporate Pantoporate Tricolporate Monocolpate Inaperturate Tricolpate Frequency (%) 24.6 19.8 13.2 7.9 14.5 3.2 16.8 Table 23: Plant sources, types and frequency of pollens obtained from Aleshinloye honey Plant species Family Pollen type Frequency (%) Parkia inundabilis Fabaceae Tricolpate 23.9 Acidanthera brevicollis Iridaceae Monocolpate 16.6 Terminalia brownii Combreataceae Heterocolporate 10.3 Gerradina foliosa Gerrardinaceae Tricolporate 14.2 Tylophora sylvatica Apocyanaceae Inaperturate 7.4 Trema guineesis Cannabaceae Diporate 27.6 Available on-line at www.afst.valahia.ro 508 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Table 24: Plant sources, types and frequency of pollens obtained from Agara honey Plant species Tilia Americana Erica arborea Celtis integrifolia Elaeis guineensis Combretum gueinzii Ipomoea ochracea Gomphrena celosioides Carya spp. Justica cordata Family Tiliaceae Ericaceae Cannabaceae Arecaceae Combretaceae Convolvulaceae Amaranthaceae Juglandaceae Acanthaceae 3.2. DISCUSSION The results of the microscopic analysis of the honey from the 24 studied areas demonstrated abundant and diversified pollen composition of the honey samples examined. The excess of bees visiting or foraging many plants may be connected to the fact that pollen is the only proteic food within the beehive; as a consequence, the pollen plays an important role in feeding the colony. In fact, pollen is used for feeding the larvae and the young bees. It contributes to body growth in general and is a determining factor in the development and the functionality of certain organs such as the adipose body, ovaries and in particular the hypopharingeal glands; these glands play an important role in Royal jelly secretion; royal jelly is used for feeding the larvae for the first three days of their life and provides the queen bee with necessary nourishment. In line with this, most of the families of plants recorded in this study have been reported to be visited by bees (Shubharani et al., 2013). It is thus shown in this study that bees visit plants regardless of their habits and habitat. The same notion was earlier reported by Adeonipekun (1989). In addition, bees prefer to visit plants with good nectar and attractive flora (Ige and Apo, 2007); though there are taxa that are non-nectariferous in nature e.g. Poaceae whose pollens are also observed in the honey samples from Apata area. Most commercially available honey is blended, meaning it is a mixture of two or more honeys differing in floral source, colour, flavour, density or geographic origin. Meanwhile, honey sample from the 16 samples without Available on-line at www.afst.valahia.ro Pollen type Tricolpate Tricolpate Triporate Monocolpate Heterocolporate Periporate Pantoporate Dicolporate Dicolporate Frequency (%) 8.7 13.5 9.6 12.4 3.5 17.1 14.5 5.3 15.4 pollens are adulterated and honey samples from the 24 pure samples are not blended but are polyfloral honey or wildflower honey i.e. derived from the nectar of many types of flowers. Generally, honey is classified by the floral source of the nectar from which it was made. Honeys can be from specific types of flower nectars or can be blended after collection. The pollen in honey is traceable to floral source and therefore region of origin (AbdulRahaman et al., 2013). In the estimation of pollen grain frequencies, the 24 pure honey samples in this work fall within the category: “secondary or accompanying pollen” (16-45%), “important minor pollen or important isolated pollen” (4 – 15% pollen) and “rare pollen or isolated pollen” (less than 3% pollen) (Vergeron, 1964). Three taxa in Apata area honey are in “secondary or accompanying pollen” ranging from (28.3% pollen frequency in Gerradinaceae -Gerradima foliosa to 15.6% in Euphorbiaceae- Tetrochidium didymostemon), four taxa are in the category “important minor pollen” and the last taxa falls to the category “minor or isolated pollen”. The pollen grain of a honey sample of Kasumu area has three taxa belonging to the category “secondary or accompanying pollen” and five taxa in the category “important minor pollen or important isolated pollen”. Pollen grains from Lafenwa honey have five taxa in “secondary or accompanying pollen” and the remaining taxa falls in the category of the “important minor pollen”. Pollen grain of honey samples from areas of Molete high school, Moor plantation, Ologede, Oremeji, Alegongo, Itoku, Iyana-cele, Majekodunmi, Basorun, Obantoko, Oluyole, 509 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 Osiele, Randa, Somaarin, Odo-eran, Sanni, Aleshinloye, and Agara has their pollen grains in the category of “secondary or accompanying pollen”, and “important minor pollen or important isolated pollen”. Pollen grains from areas of the University of Ibadan, Atinsola, and Crescent University have their pollen grains in the category: “secondary or accompanying pollen”, “important minor pollen or important isolated pollen” and “minor or isolated pollen”. The number of granules examined depends on the degree of precision required when estimating the PK (pollen grain) frequencies. For an indicative sample evaluation, the computation of about 100 PK (PK = pollen grain) should be sufficient. It is necessary to count 200-300 PK to determine the frequency classes. For a precise percentage calculation 1000-1200 PK have to be counted (Vergeron, 1964). Thus the honey samples were considered rich, poor and extremely poor in number of pollen grain per 1gm of honey samples if 1200, 1000 and below 1000 pollens were counted. This is a modification to Maurizio (1975). Based on this, the pollen grains of Gerradina foliosa in Apata honey, Tilia americana in Kasumu honey, Acidanthera brevicollis and Piptadenia africana in Lafenwa honey, Typha capensis and Typha augustifolia in Molete High School honey, Clausena anisata in Moor plantation honey, Berlinia bifoliata Hymenocardia acida in Ologede honey, Acidanthera brevicollis, Hymenocardia acida, and Lannea acida in Oremeji honey, Typha capensis and Turraeanthus africana in University of Ibadan honey, Elaeis guineensis in Alegongo honey, Alchornea cordifolia and Hymenocardia acida in Atinsola honey, Piptadenia africana in Itoku honey, Vitex doniana and Acridocarpus macrocalyx in Iyana-cele honey, Alchornea cordifolia and Indigofera leptoclada in Obantoko honey, Agrimonia eupatoria and Piptadenia africana in Oluyole honey, Celtis integrifolia in Randa honey, Acridocarpus macrocalyx in Sanni honey and Parkia inundabilis Trema guineensis in Aleshinloye honey are considered rich and produced rich pollens. Available on-line at www.afst.valahia.ro 4. CONCLUSIONS The pollen composition of the honey samples in this study has shown that honeybees travel a considerable distance in search of suitable food materials (nectar) for their survival and production of honey. The presence of the pollen grains in the 24 honey samples is a clear indication that the honeys are not adulterated but pure and they are not unifloral but multifloral, and the absence of pollen grains in the remaining 16 honey samples is an indication that the honey samples are adulterated and not pure. 5. REFERENCES [1] Abdulla, F. and Abdulaziz, M. A. (1998). The prophylactic and curative effect of cedar honey induced ulcers in rabbits. The Second International Arab Apicultural Conference- Amman, 1:26-31. [2] Abdulrahaman, A. A., Solomon, O. R., Adeyemi, S. B., Liadi, M. T., Ahmed, R. N., Belewu, M. A., and Oladele, F. A., (2013). Melisopalynological analysis of honey samples from Jatropha Plantation and Unilorin Apiary Farm. International Journal of Phytofuels and Allied Sciences, 2(1): 8192. [3] Adeonipekun, P. A. (1989). A Palynological Study of an Apiary in Ibadan, Nigeria, Unpublished Report for B.Sc. (Hons), Department of Botany, University of Ibadan, Nigeria. [4] Bryant, V. and Jones, S. K. (2001). The r-values of honey: pollen coefficients. Palynology, 25(1): 1128. [5] Dieter, D. A., Pavel, E., Tarasov A., Takeshi N. B., and Suigetsu, A. N., (2013). Atlas of pollen, spores and further non-pollen palynomorphs recorded in the glacial-interglacial late Quaternary sediments of Lake Suigetsu, central Japan. Quaternary International, 290-291: 164-238. [6] Erdtman, G. (1960). The acetolysis method. A revised description. Svensk Botany Tidskr., 51: 561567. [7] Ige, O. E. and Apo, K. A. (2007). Pollen analysis of honey samples from two vegetation zones in Nigeria. Science Focus, 13: 36-43. [8] Jusbin, O. S. (1996). Chemical Composition and Application. In: Schmidt (Ed) Bee Products. Plenum Press, New York: 25- 26pp. [9] Louveaux, J., Maurizio, A. and Vorwohl, G. (1978). Methods of melissopalynology. Bee World, 59: 39-157. [10] Maurizio, A. (1951). Pollen analysis of honey. Bee World, 32: 1 – 5. 510 Volume 18, Issue 3, 2017 Annals. Food Science and Technology 2017 [11] Maurizio, A. (1975). Microscopy of honey. In Crane, E. (ed) Honey: a comprehensive survey. Heinemann; London, UK; pp 240-257. [12] Molan, P. C. (1998). The limitations of the methods of identifying the floral source of honeys. Bee World 79: 59 – 68. [13] Molan, P. C. (2001). The Potential of honey to promote oral wellness. General Dentistry: 586: 5. [14] Shubharani, R., Roopa, P. and Sivaram, V. (2013). Pollen morphology of selected bee forage plants. Global Journal of Bio-Science and Biotechnology, 2 (1): 82-90. [15] Terrab, A., Diez M. J. and Heredia, F. J. (2003). Palynological, physicochemical and colour characterisation of Moroccan honeys. River red gum (Eucalyptus camaldulensis Dehnl.) honey. Available on-line at www.afst.valahia.ro [16] [17] [18] [19] 511 International Journal of Food Science and Technology, 38: 379-386. Vergeron, P. (1964). Statistical interpretation and analysis of pollen. Ann. Abeille, 7 (4): 349-364. Wahdan, H. (1998). Causes of the antimicrobial activity of honey. Infection, 26 (1): 26–31. William, D. G., Charlotte, S. M. and Daniel, A. L (2013). Atlas of the tropical West African pollen flora. Review of Palaeobotany and Palynology, 199: 1–135. Zet-Sche, B. (1932). Pollen grains. Review of Paleobotany and Palynology, 20: 151-160. Volume 18, Issue 3, 2017