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INTRODUCTION
The pollen collected by honey bees (Apis mellifera) has a high
nutritive value and is composed of approximately 40% carbohy-
drates, 35% proteins, 5% water, 5% lipids and from five to 15%
other substances. In many parts of the world pollen is used as a
human dietary supplement. Recommendations for daily con-
sumption 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, 1997). Orzaez et al.
(2002) studied the composition and caloric value of 15 pollen
samples.
The ratio of unsaturated/saturated fatty acids in pollen present-
ed by several authors (Bonvehi et al., 1986; Bonvehi & Jordá,
1997; Loper et al., 1980) is higher than 1.0, 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 botan-
ical origin.
MATERIALS AND METHODS
Fourteen pollen samples were acquired between 1999 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 –10 °C until the moment of analysis. Sam-
ples were ground on an ARNO (Brazilian trademark for house-
hold 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, 1959). 100 mg were con-
verted into fatty acid methyl esters with the esterifying reagent
BF3-methanol following the official method of the American Oil
Chemist’s Society (AOCS, 1993). After the derivation, the
methyl esters dissolved in hexane were analysed by gas chro-
matography.
Determining the fatty acid composition by gas
chromatography
The methyl esters of fatty acids were analysed by gas chro-
matography in a CHROMPACK chromatographer, model CP
9001with a flame ionization detector (FID) and a capillary col-
umn (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 180 °C, for 7 min. The chromatographer was
programmed for 5 °C/min, reaching a maximum temperature of
220 °C. The carrier gas was hydrogen (H2) 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 affect-
ed areas by Chromato-Integrator and expressed in relative per-
centages.
Pollen load analysis
Ten pollen loads of each sample were macerated in 10 ml of 50%
alcohol, left in maceration for 15 minutes and centrifuged for
*Corresponding author: dmbastos@usp.br
Journal of Apicultural Research 43(2): 35–39 (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
D
EBORAH
H M
ARKOWICZ
B
ASTOS
,11* O
RTRU D
M
ONIKA
B
ARTH
,2C
ÁSSIA
I
SABEL
R
OCHA
,3I
LDENIZE
B
ARBOSA DA
S
ILVA
C
UNHA
,3P
ATRICIA DE
O
LIVEIRA
C
ARVALHO
,3E
LIZABETH
A S
ILVA
T
ORRES
11
AND
M
ARCOS
M
ICHELAN
1Nutrition Department, School of Public Health, São Paulo University, Av. Dr. Arnaldo, 715. CEP 01246-904 São Paulo, Brazil
2Department of Virology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro and Laboratory of Palinology, Biology Institute,
Universidade Federal do Rio de Janeiro, Brazil
3School of Pharmacy, São Francisco University, Av. São Francisco de Assis, 218. Caixa Postal 163. CEP 12916-900. Bragança
Paulista, SP, Brazil
Received 6 June 2003, accepted subject to revision 3
1
October 2003, accepted for publication
1
5 March 2004
SUMMARY
The fatty-acid composition and botanical origin of 14 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 18.6% 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
10–15 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 centrifu-
gation and decantation, the tubes were kept upside down for 10
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 consid-
ered which may correspond to various taxa that present the
same or similar pollen morphology. The pollen identification was
based on Barth (1989).
RESULTS
The fatty acids occurring in greatest proportion in all analysed
pollen samples (table 1) are: oleic acid (C18:1), linoleic acid
(C18:2) and arachidic acid (C20:0). Only one sample did not con-
tain palmitic acid (C16:0), which was present in significant pro-
portions (13.5% to 27.8%) in the other samples (table 1).
Linolenic acid (C18:2) was found in a bifloral (A18) and a hetero-
floral (A20) pollen sample, showing contribution from the Are-
caceae, Asteraceae, Brassicaceae and Polygonaceae families.
Tables 1and 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 vari-
able. There are considerable variations in the unsaturated/satu-
rated fatty acid ratio which may happen due to the botanical ori-
gin or to the processing and storage conditions.
The botanical origin of the samples is presented in table 3. Twen-
ty-eight taxa could be identified. Three samples can be consid-
ered as unifloral loads: sample A13 of Eucalyptus spp. (Myrtaceae)
pollen grains, A17 of Arecaceae pollen grains and A19 of Aster-
aceae pollen grains, Eupatorium spp. type. Six samples are of biflo-
ral loads, sample A18 presenting pollen grains of Brassicaceae
and Antigonon leptopus (Polygonaceae), sample A22 of the Myr-
cia 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 Eupa-
torium spp. types and A30 of Antigonon leptopus and Eupatorium
spp. types. Five samples are multifloral loads from more than
two taxa (A11, A12, A14, A20, A21) from which the most rep-
resentative 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 unsat-
urated fatty acids between 18.6 and 55.9% of the total fatty acids.
Three unifloral pollen samples (A13, A17, A19), of different
36 Bastos, Barth, Rocha, Cunha, Carvalho, Torres; Michelan
TABLE 11. Percentage fatty acid composition (%) of pollen samples from Minas Gerais and São Paulo, Brazil,
collected between 11999 and 2000. Tr = trace.
Samples C8:0 C112:0 C114:0 C116:0 C118:0 C118:11C118:2 C118:3 C20:0 C22:0
caprylic lauric myristic palmitic stearic oleic linoleic linolenic arachidic behenic
A11 Tr Tr Tr 22.7 4.5 8.2 21.3 Tr 31.2 Tr
A123.4 01.3 Tr 23.8 Tr 6.5 16.4 Tr 15.9 Tr
A133.0 Tr 3 24.0 1.9 7.2 32.6 Tr 15.7 Tr
A14Tr27.9 Tr Tr 3.0 8.6 19.8 Tr 31.12.8
A17TrTr1.2 27.8 Tr 3.9 28.4 Tr 18.8 1.9
A18TrTrTr21.8 Tr 5.5 09.3 3.8 42.7 Tr
A194.8 Tr 6.4 27.5 Tr 8.125.6 Tr 21.1Tr
A20 Tr Tr Tr 2 6 . 1Tr 9.6 24.11.4 25.7 3.3
A21Tr Tr Tr 13.5 3.6 7.7 29.5 Tr 20.3 Tr
A22 Tr Tr Tr 26.3 3.4 7.2 31.4 Tr 21.6 2.0
A24 Tr Tr Tr 27.8 3.19.9 21.2 Tr 28.5 3.7
A25 Tr Tr 1.3 21.0 Tr 4.9 08.9 Tr 24.9 Tr
A26 Tr Tr Tr 2 1.0 1.9 6.2 49.7 Tr 08.9 Tr
A30 Tr Tr 1.8 26.3 2.0 5.8 13.3 Tr 22.3 Tr
Max value 4.8 27.9 6.4 27.8 4.5 9.9 49.7 3.8 42.7 3.7
Min value Tr Tr 1.2 Tr Tr 3.9 08.9 Tr 08.9 Tr
TABLE 2. Range of fatty acid content in bee
Reference11n2Fatty acid
caprylic decanoic lauric myristic myristoleic palmitic
1317.3–0.5 ––– 4.7–0.9 6.9–0.4 3.2–0.0 36.1–15.3
25––– 0.9–0.0 5.7–0.2 39.9–11.5 3.8–0.0 28.3–14.9
3201.8–0.15.3–0.9 3.5–0.9 4.1–0.5 1.7–0.133.7–18.5
This work 144.8–Tr ––– 27.9–Tr 6.4–1.2 ––– 27.8–Tr
11Boenvehi et al.(1986), 2 Shawer et al.(1987), 3 Bonvehi & Jordá (1997)
2n= number of samples analysed
botanical origins, present a high level of unsaturated fatty acids
(more than 32%). Among the bifloral samples, those which con-
tain pollen of Antigonon leptopus (A18, 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 (A13, A19, A26) present a high percent-
age. The multifloral samples, A20 and A21, both containing
pollen from the Arecaceae family, present high levels of unsatu-
rated fatty acids.
DISCUSSION
Numerous studies around the world have characterized the fatty
acid composition of bee-collected pollens (Bonvehi et al., 1986;
Bonvehi & Jordá, 1997; Farag et al., 1978; Loper et al., 1980; Van
der Vorst et al., 1982; Shawer et al., 1987; Youssef et al., 1978).
A comparison of these data is on table 2. Pollen grains collect-
ed by bees, especially when in contact with bee saliva are sub-
mitted to several changes such as the oxidation of unsaturated
fatty acids by the action of hydrolytic enzymes in the saliva, tem-
perature, and exposure to oxygen. In addition, Apis-collected
pollen is manipulated by beekeepers using dehydration proce-
dures which may be harmful to the unsaturated fatty acids.
The ratio of unsaturated to saturated fatty acids found in this
study was < 1.0 in almost all samples (table 4). Unsaturated/sat-
urated fatty acid ratio varied from 0.6 to 1.0 in unifloral pollen,
while multifloral samples showed unsaturated/saturated ratios
from 0.4 to 0.8 and bifloral pollen from 0.3 to 1.7. This may have
happened due to the composition of the pollen load or may have
been the result of inadequate processing practices of this mate-
rial, mainly during the dehydration procedure or storage condi-
tions (light and temperature). As bees usually select pollen with
a high level of unsaturated fatty acids (Bonvehi & Jordá, 1997), it
seems that the unsaturated fatty acid degradation during manip-
ulation/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 lep-
topus and the pollen types of Croton, Gochnatia, Ilex, Montanoa,
Senecio and Vernonia spp. (table 3). Pollen loads of other botan-
ical taxa occurred in low percentages and in no case did the bi-
and multifloral samples present the same pollen spectrum.
Plant species of native vegetation predominated, including trees
such as palms (Arecaceae), some Asteraceae (Gochnatia, Mon-
tanoa spp.) and Croton spp. (Euphorbiaceae), and shrubs like Ver-
nonia spp, and herbs (Eupatorium, Senecio spp.). Other taxa, such
as Brassicaceae, Cupressaceae, Ilex spp. and Senecio spp. (Aster-
aceae) 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 (1997)
in the Bananal region bordering the states of São Paulo and Rio
de Janeiro, and by Barth & Luz (1998) in a mangrove area near
Rio de Janeiro.
These results bring new information on pollen fatty acid com-
position as well as the botanical sources visited by pollen-col-
lecting bees. The data will help regulatory bodies establish
parameters of quality control for pollen produced in Brazil. Fur-
ther studies are necessary to discriminate the effects of botan-
ical 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).
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151pp.
BARTH, O M; LUZ, C F P (1998) Melissopalynological data obtained from a mangrove
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acteristicas fisico-quimicas del polen de abejas. Alimentaria 176: 63–67.
BONVEHI, J S; JORDÁ, R E (1997) Nutrient composition and microbiological quality
of honeybee-collected pollen in Spain. Journal of Agricultural and Food Chem-
istry 45: 725–732.
DUTRA, V M; BARTH, O M (1997) Análise palinológica de amostras de mel da região
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Número especial: 174–183.
FARAG, R S; YOUSSEF, A M; EWIES, M A; HALLABO, S A S (1978) Long-chain fatty
acids of six pollens collected by honeybees in Egypt. Journal of Apicultural
Research 17(2): 100–104.
LINSKENS, H F; JORDE, W (1997) Pollen as food and medicine – a review. Economic
Botany 51: 78–87.
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SHAWER, M B; ALI, S M; ABDELLATIF, M A; EL-REFAI, A A (1987) Biochemical stud-
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VORST, E V
VAN DER
; MATTYS, J; RYCKE P, H; JACOBS, F J (1982) Comparative lipid
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Fatty acid composition and botanical origin of bee pollen loads from Brazil 37
e-collected pollen described in the literature.
palmitoleic stearic oleic linoleic linolenic arachidic behenic
5.0–0.0 7.2–0.4 18.6–8.0 41.6–10.6 25.3–3.3 4.2–0.0 2.7–0.0
––– 10.1–2.9 58.0–3.6 12.5––0.0 29.9–0.0 1.6–0.0 –––
5.05–0.14.0–0.3 19.0–8.7 41.6–10.6 25.3–11.7 1.0–0.12.6–0.1
––– 4.5–Tr 9.9–3.9 49.7–8.9 3.8–Tr 42.7–8.9 3.7–Tr
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
11999 and 2000. (+++) = very frequent pollen grains; (++) = frequent pollen grains; (+) = few pollen grains;
(rare) = very few pollen grains.
Samples Identified taxa Frequency Characterization
A11 Asteraceae: Eupatorium type +++ multifloral
Asteraceae: Artemisia type ++
Asteraceae: Vernonia type ++
Euphorbiaceae: Croton ++
Myrtaceae: Eucalyptus +
Verbenaceae: Lantana +
not identified +
A12Mimosaceae: Mimosa verrucosa type +++ multifloral
Asteraceae: Elephantopus type ++
Asteraceae: Gochnatia type ++
Asteraceae: Senecio type +
Euphorbiaceae: Croton +
Myrtaceae: Eucalyptus +
A13Myrtaceae: Eucalyptus +++ unifloral
Asteraceae: Montanoa type +
A14Arecaceae +++ multifloral
Poaceae +++
Asteraceae: Montanoa type ++
Cyperaceae +
Monocotiledonea +
Loranthaceae rare
A17Arecaceae +++ unifloral
Aquifoliaceae: Ilex +
Bignoniaceae (?) +
A18Brassicaceae +++ bifloral
Polygonaceae: Antigonon leptopus ++
Asteraceae +
Bignoniaceae (?) +
A19Asteraceae: Eupatorium type +++ unifloral
Asteraceae: Senecio type rare
A20 Arecaceae ++ multifloral
Asteraceae: Eupatorium type ++
Not identified ++
Asteraceae: Senecio type +
Myrtaceae: Eucalyptus +
Bombacaceae rare
Poaceae rare
A21Myrtaceae: 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 rare
Cupressaceae rare
A26 Myrtaceae: Eucalyptus +++ bifloral
Asteraceae: Eupatorium type ++
Commelinaceae: Commelina rare
Combretaceae/Mestomataceae rare
A30 Polygonaceae: Antigonon leptopus +++ bifloral
Asteraceae: Eupatorium type +++
Brassicaceae +
Poaceae: Zea mays rare
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 11999 and 2000.11
Botanical characterization Sample Saturated fatty Unsaturated fatty Unsaturated/saturated
acids (%) acids (%) ratio
Multifloral A11 58.4 29.5 0.5
Multifloral A1244.4 22.9 0.5
Multifloral A1464.8 28.4 0.8
Multifloral A20 55.135.10.6
Multifloral A2137.4 37.2 0.3
Unifloral A1347.6 39.8 0.6
Unifloral A1959.8 33.7 0.6
Unifloral A1749.7 32.3 1.0
Bifloral A1864.5 18.6 0.4
Bifloral A22 53.3 38.6 0.7
Bifloral A24 63.131.10.5
Bifloral A25 47.2 13.8 0.3
Bifloral A26 31.8 55.9 1.7
Bifloral A30 52.4 19.10.4
1Sum of fatty acids present at more than 1% level
