Fatty acid composition and palynological analysis of bee (Apis) pollen loads in the states of São Paulo and Minas Gerais, Brazil

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1 Journal of Apicultural Research 43(2): (2004) IBRA 2004 ORIGINAL ARTICLE Fatty acid composition and palynological analysis of bee (Apis) pollen loads in the states of São Paulo and Minas Gerais, Brazil DEBORAH H MARKOWICZ BASTOS, * ORTRUD MONIKA BARTH, 2 CÁSSIA ISABEL ROCHA, 3 ILDENIZE BARBOSA DA SILVA CUNHA, 3 PATRICIA DE OLIVEIRA CARVALHO, 3 ELIZABETH A SILVA TORRES AND MARCOS MICHELAN Nutrition Department, School of Public Health, São Paulo University, Av. Dr. Arnaldo, 75. CEP São Paulo, Brazil 2 Department of Virology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro and Laboratory of Palinology, Biology Institute, Universidade Federal do Rio de Janeiro, Brazil 3 School of Pharmacy, São Francisco University, Av. São Francisco de Assis, 28. Caixa Postal 63. CEP Bragança Paulista, SP, Brazil Received 6 June 2003, accepted subject to revision 3 October 2003, accepted for publication 5 March 2004 SUMMARY The fatty-acid composition and botanical origin of 4 honey bee (Apis mellifera) pollen load samples acquired in shops and apiaries in Minas Gerais and São Paulo states, Brazil, were determined. The fatty acids presented a variable composition among these samples. All samples contained oleic, linoleic and arachidic acid. Only one sample did not contain palmitic acid. The unsaturated fatty-acid level varied from 8.6% to 55.9% of the total fatty-acid composition, suggesting that pollen is a good source of unsaturated fatty acids to the diet. Several pollen types were identified. Eucalyptus (Myrtaceae) and Eupatorium (Asteraceae) pollen types were the most common among those sampled. These data might help the regulatory agencies establish quality parameters for pollen produced in Brazil. There are no additional data available on Brazilian pollen fatty acid composition. Keywords: pollen, fatty acid composition, botanical origin, Brazil INTRODUCTION The pollen collected by honey bees (Apis mellifera) has a high nutritive value and is composed of approximately 40% carbohydrates, 35% proteins, 5% water, 5% lipids and from five to 5% other substances. In many parts of the world pollen is used as a human dietary supplement. Recommendations for daily consumption of pollen varies from one to three teaspoons (5 25 g) per day, mixed or not mixed with other food such as yogurt, honey, fruit juice etc. (Linskens & Jorde, 997). Orzaez et al. (2002) studied the composition and caloric value of 5 pollen samples. The ratio of unsaturated/saturated fatty acids in pollen presented by several authors (Bonvehi et al., 986; Bonvehi & Jordá, 997; Loper et al., 980) is higher than.0, supporting the use of bee pollen as a food supplement, rich in unsaturated fatty acids. There are no studies related to the fatty acid composition of pollen load samples collected in Brazil, a country in which pollen is regularly recommended and consumed as a food supplement. The present study aims to provide new information about fatty acid composition of pollen loads collected by bees in the states of São Paulo and Minas Gerais, Brazil, and to verify their botanical origin. MATERIALS AND METHODS Fourteen pollen samples were acquired between 999 and 2000 in several regions of Minas Gerais and São Paulo states, Brazil, at local businesses and apiaries. All samples were classified as dehydrated pollen loads of Apis mellifera bees. The samples were stored in a freezer at 0 C until the moment of analysis. Samples were ground on an ARNO (Brazilian trademark for household appliances) multi-processor, model PRO, and shaken through a 24 sieve (adhering to ASTM standards) in preparation for chemical analyses. Preparation of the fatty acid methyl esters The lipids were extracted from the samples following Bligh- Dyer s modified method (Bligh & Dyer, 959). 00 mg were converted into fatty acid methyl esters with the esterifying reagent BF 3 -methanol following the official method of the American Oil Chemist s Society (AOCS, 993). After the derivation, the methyl esters dissolved in hexane were analysed by gas chromatography. Determining the fatty acid composition by gas chromatography The methyl esters of fatty acids were analysed by gas chromatography in a CHROMPACK chromatographer, model CP 900 with a flame ionization detector (FID) and a capillary column (CP-Sil 88 50m 0.25 mm). The detector temperature was 280 C; the injector was 250 C and the initial temperature of the column was 80 C, for 7 min. The chromatographer was programmed for 5 C/min, reaching a maximum temperature of 220 C. The carrier gas was hydrogen (H 2 ) with a flow rate of 2.0 ml/min at the column. Identification of the fatty acids was made by comparing the retention time of the sample compounds with those for standards injected in the same conditions. The standard spiking technique was also used along with the sample. Quantification was performed by the normalization of the affected areas by Chromato-Integrator and expressed in relative percentages. Pollen load analysis Ten pollen loads of each sample were macerated in 0 ml of 50% alcohol, left in maceration for 5 minutes and centrifuged for *Corresponding author: dmbastos@usp.br

2 36 Bastos, Barth, Rocha, Cunha, Carvalho, Torres; Michelan 0 5 min. Ten ml of distilled water and one drop of absolute alcohol were added to the sediment and centrifuged again. In sequence, a mixture of equal parts of glycerin and distilled water were added to the sediment and left for 30 min. After centrifugation and decantation, the tubes were kept upside down for 0 min. The sediment was then added to non-stained glycerine jelly. The slides were sealed with paraffin. The pollen grains were compared for taxonomic identification with a reference slide collection. When the identified botanical taxon was above the species level, the pollen type was considered which may correspond to various taxa that present the same or similar pollen morphology. The pollen identification was based on Barth (989). RESULTS The fatty acids occurring in greatest proportion in all analysed pollen samples (table ) are: oleic acid (C8:), linoleic acid (C8:2) and arachidic acid (C20:0). Only one sample did not contain palmitic acid (C6:0), which was present in significant proportions (3.5% to 27.8%) in the other samples (table ). Linolenic acid (C8:2) was found in a bifloral (A8) and a heterofloral (A20) pollen sample, showing contribution from the Arecaceae, Asteraceae, Brassicaceae and Polygonaceae families. Tables and 2 show that qualitative composition of fatty acids of Apis-collected pollen is basically the same among the studied samples. However, the proportion of those compounds is variable. There are considerable variations in the unsaturated/saturated fatty acid ratio which may happen due to the botanical origin or to the processing and storage conditions. The botanical origin of the samples is presented in table 3. Twenty-eight taxa could be identified. Three samples can be considered as unifloral loads: sample A3 of Eucalyptus spp. (Myrtaceae) pollen grains, A7 of Arecaceae pollen grains and A9 of Asteraceae pollen grains, Eupatorium spp. type. Six samples are of bifloral loads, sample A8 presenting pollen grains of Brassicaceae and Antigonon leptopus (Polygonaceae), sample A22 of the Myrcia spp. type (Myrtaceae) and Ilex spp. (Aquifoliaceae), sample A24 of Myrcia spp. type and Arecaceae, sample A25 of Antigonon leptopus and Brassicaceae, sample A26 of Eucalyptus and Eupatorium spp. types and A30 of Antigonon leptopus and Eupatorium spp. types. Five samples are multifloral loads from more than two taxa (A, A2, A4, A20, A2) from which the most representative are the Eupatorium spp. type, Mimosa verrucosa type (Mimosaceae), Arecaceae and Poaceae. (A pollen type may include several genera or species, the pollen grains of which present similar morphology). Table 4 shows that the analysed samples present a level of unsaturated fatty acids between 8.6 and 55.9% of the total fatty acids. Three unifloral pollen samples (A3, A7, A9), of different TABLE. Percentage fatty acid composition (%) of pollen samples from Minas Gerais and São Paulo, Brazil, collected between 999 and Tr = trace. Samples C8:0 C2:0 C4:0 C6:0 C8:0 C8: C8:2 C8:3 C20:0 C22:0 caprylic lauric myristic palmitic stearic oleic linoleic linolenic arachidic behenic A Tr Tr Tr Tr 3.2 Tr A Tr 23.8 Tr Tr 5.9 Tr A3 3.0 Tr Tr 5.7 Tr A4 Tr 27.9 Tr Tr Tr A7 Tr Tr Tr Tr A8 Tr Tr Tr 2.8 Tr Tr A9 4.8 Tr Tr Tr 2. Tr A20 Tr Tr Tr 26. Tr A2 Tr Tr Tr Tr 20.3 Tr A22 Tr Tr Tr Tr A24 Tr Tr Tr Tr A25 Tr Tr Tr Tr 24.9 Tr A26 Tr Tr Tr Tr 08.9 Tr A30 Tr Tr Tr 22.3 Tr Max value Min value Tr Tr.2 Tr Tr Tr 08.9 Tr TABLE 2. Range of fatty acid content in bee Reference n 2 Fatty acid caprylic decanoic lauric myristic myristoleic palmitic This work Tr 27.9 Tr Tr Boenvehi et al.(986), 2 Shawer et al.(987), 3 Bonvehi & Jordá (997) 2 n = number of samples analysed

3 Fatty acid composition and botanical origin of bee pollen loads from Brazil 37 botanical origins, present a high level of unsaturated fatty acids (more than 32%). Among the bifloral samples, those which contain pollen of Antigonon leptopus (A8, A25, A30) present low relative percentage values (lower than 20%) of unsaturated fatty acids, while the samples containing pollen of Eucalyptus and Eupatorium spp. types (A3, A9, A26) present a high percentage. The multifloral samples, A20 and A2, both containing pollen from the Arecaceae family, present high levels of unsaturated fatty acids. DISCUSSION Numerous studies around the world have characterized the fatty acid composition of bee-collected pollens (Bonvehi et al., 986; Bonvehi & Jordá, 997; Farag et al., 978; Loper et al., 980; Van der Vorst et al., 982; Shawer et al., 987; Youssef et al., 978). A comparison of these data is on table 2. Pollen grains collected by bees, especially when in contact with bee saliva are submitted to several changes such as the oxidation of unsaturated fatty acids by the action of hydrolytic enzymes in the saliva, temperature, and exposure to oxygen. In addition, Apis-collected pollen is manipulated by beekeepers using dehydration procedures which may be harmful to the unsaturated fatty acids. The ratio of unsaturated to saturated fatty acids found in this study was <.0 in almost all samples (table 4). Unsaturated/saturated fatty acid ratio varied from 0.6 to.0 in unifloral pollen, while multifloral samples showed unsaturated/saturated ratios from 0.4 to 0.8 and bifloral pollen from 0.3 to.7. This may have happened due to the composition of the pollen load or may have been the result of inadequate processing practices of this material, mainly during the dehydration procedure or storage conditions (light and temperature). As bees usually select pollen with a high level of unsaturated fatty acids (Bonvehi & Jordá, 997), it seems that the unsaturated fatty acid degradation during manipulation/storage can explain the results obtained in the present work. Unfortunately, there are no additional data on Brazilian fatty acids pollen composition that would ratify this assumption. In relation to the pollen taxonomic spectra, the samples from Minas Gerais and São Paulo states represented Eucalyptus spp. (several species introduced to Brazil) and Eupatorium spp. as the main pollen types. Additionally, pollen from well-known bee plant species was found in smaller percentages, such as Antigonon leptopus and the pollen types of Croton, Gochnatia, Ilex, Montanoa, Senecio and Vernonia spp. (table 3). Pollen loads of other botanical taxa occurred in low percentages and in no case did the biand multifloral samples present the same pollen spectrum. Plant species of native vegetation predominated, including trees such as palms (Arecaceae), some Asteraceae (Gochnatia, Montanoa spp.) and Croton spp. (Euphorbiaceae), and shrubs like Vernonia spp, and herbs (Eupatorium, Senecio spp.). Other taxa, such as Brassicaceae, Cupressaceae, Ilex spp. and Senecio spp. (Asteraceae) suggest as sites of pollen origin the south-east/southern regions of Brazil. The great diversity of botanical species in the bee pollen loads is consistent with observations made by Dutra & Barth (997) in the Bananal region bordering the states of São Paulo and Rio de Janeiro, and by Barth & Luz (998) in a mangrove area near Rio de Janeiro. These results bring new information on pollen fatty acid composition as well as the botanical sources visited by pollen-collecting bees. The data will help regulatory bodies establish parameters of quality control for pollen produced in Brazil. Further studies are necessary to discriminate the effects of botanical origin versus processing and storage conditions on pollen unsaturated/saturated fatty acid ratios. Acknowledgements Financial support for botanical investigations was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). REFERENCES AMERICAN OIL CHEMIST S SOCIETY (993) Official methods and recommended practices of the American Oil Chemists Society. Champaign, v 2 (4th edition). BARTH, O M (989) O Pólem no mel Brasileiro. Gráfica Luxor; Rio de Janeiro, Brazil; 5 pp. BARTH, O M; LUZ, C F P (998) Melissopalynological data obtained from a mangrove area near to Rio de Janeiro, Brazil. Journal of Apicultural Research 37: BLIGH, E G; DYER, J W (959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37(8): BONVEHI, J S; GALINDO, J G; PAJUELO, A G (986) Estudio de la composición y caracteristicas fisico-quimicas del polen de abejas. Alimentaria 76: BONVEHI, J S; JORDÁ, R E (997) Nutrient composition and microbiological quality of honeybee-collected pollen in Spain. Journal of Agricultural and Food Chemistry 45: DUTRA, V M; BARTH, O M (997) Análise palinológica de amostras de mel da região de Bananal (SP/RJ), Brasil. Revista Universidade de Guarulhos, GeociÉncias II, Número especial: FARAG, R S; YOUSSEF, A M; EWIES, M A; HALLABO, S A S (978) Long-chain fatty acids of six pollens collected by honeybees in Egypt. Journal of Apicultural Research 7(2): LINSKENS, H F; JORDE, W (997) Pollen as food and medicine a review. Economic Botany 5: LOPER, G M; STANDIFER, L N; THOMPSON, M J; GILLIAM, M (980) Biochemistry and microbiology of bee-collected almond (Prunus dulcis) pollen and bee bread I Fatty acids, vitamins and minerals. Apidologie (): ORZAEZ, V M T; DIAZ, M A; BRAVO, S R; BELLAN, G B (2002) The importance of bee collected pollen in the diet: a study of its composition. International Journal of Food Science and Nutrition 53(3): SHAWER, M B; ALI, S M; ABDELLATIF, M A; EL-REFAI, A A (987) Biochemical studies of bee-collected pollen in Egypt 2 Fatty acids and non-saponifiables. Journal of Apicultural Research 26(2): 33 36, 987. VORST, E V VAN DER; MATTYS, J; RYCKE P, H; JACOBS, F J (982) Comparative lipid composition of colony and laboratory-stored pollen. Journal of Apicultural Research 2(3): YOUSSEF, A M; FARAG, R S; EWIES, M A.; EL-SHAKA, A S M A (978) Chemical studies on pollen collected by honey bees in Giza region, Egypt. Journal of Apicultural Research 7: collected pollen described in the literature. palmitoleic stearic oleic linoleic linolenic arachidic behenic Tr Tr Tr

4 38 Bastos, Barth, Rocha, Cunha, Carvalho, Torres; Michelan TABLE 3. Analysis of pollen load samples from Minas Gerais and São Paulo states, Brazil, collected between 999 and (+++) = very frequent pollen grains; (++) = frequent pollen grains; (+) = few pollen grains; () = very few pollen grains. Samples Identified taxa Frequency Characterization A Asteraceae: Eupatorium type +++ multifloral Asteraceae: Artemisia type ++ Asteraceae: Vernonia type ++ Euphorbiaceae: Croton ++ Verbenaceae: Lantana + not identified + A2 Mimosaceae: Mimosa verrucosa type +++ multifloral Asteraceae: Elephantopus type ++ Asteraceae: Gochnatia type ++ Asteraceae: Senecio type + Euphorbiaceae: Croton + A3 ++ unifloral Asteraceae: Montanoa type + A4 Arecaceae +++ multifloral Poaceae +++ Asteraceae: Montanoa type ++ Cyperaceae + Monocotiledonea + Loranthaceae A7 Arecaceae +++ unifloral Aquifoliaceae: Ilex + Bignoniaceae (?) + A8 Brassicaceae +++ bifloral Polygonaceae: Antigonon leptopus ++ Asteraceae + Bignoniaceae (?) + A9 Asteraceae: Eupatorium type +++ unifloral Asteraceae: Senecio type A20 Arecaceae ++ multifloral Asteraceae: Eupatorium type ++ Not identified ++ Asteraceae: Senecio type + Bombacaceae Poaceae A2 Myrtaceae: Myrcia type +++ multifloral Aquifoliaceae: Ilex ++ Arecaceae ++ A22 Myrtaceae: Myrcia type +++ bifloral Aquifoliaceae: Ilex ++ Arecaceae + A24 Myrtaceae: Myrcia type +++ bifloral Arecaceae ++ Asteraceae: Eupatorium type + Aquifoliaceae: Ilex spp. Rare A25 Polygonaceae: Antigonon leptopus +++ bifloral Fabaceae ++ Brassicaceae + Chenopodiaceae Cupressaceae A26 ++ bifloral Asteraceae: Eupatorium type ++ Commelinaceae: Commelina Combretaceae/Mestomataceae A30 Polygonaceae: Antigonon leptopus +++ bifloral Asteraceae: Eupatorium type +++ Brassicaceae + Poaceae: Zea mays

5 Fatty acid composition and botanical origin of bee pollen loads from Brazil 39 TABLE 4. Percentage composition of saturated and unsaturated fatty acids of pollen samples from Minas Gerais and São Paulo, Brazil, collected between 999 and Botanical characterization Sample Saturated fatty Unsaturated fatty Unsaturated/saturated acids (%) acids (%) ratio Multifloral A Multifloral A Multifloral A Multifloral A Multifloral A Unifloral A Unifloral A Unifloral A Bifloral A Bifloral A Bifloral A Bifloral A Bifloral A Bifloral A Sum of fatty acids present at more than % level