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Melissopalynology is the study of flora and origin of honey samples using pollen that is collected and trapped in the honey.
November 2018 BEE CULTURE 41
The Science Of Using Pollen To Study Honey
Vaughn Bryant
Melissopalynology is the study of pollen in honey. The
term comes from the Greek words for “bee” and “honey”
along with the words for “study of dust,” which now refers
to “pollen.” Today, it is recognized worldwide as being the
least expensive and quickest way to determine the oral
contents and geographical origin of honey. However, the
effectiveness of the technique depends on the skills of the
pollen analysis (palynologist), the method of extracting the
pollen from honey samples, and the skill of the analyst
in interpreting the results. Today honey has become an
important commercial business and provides sweetness
used in thousands of products.
Humans have ve basic taste abilities, sweet, sour,
bitter, salty, and umami (de ned as savory). Of these, we
generally do not enjoy foods that are too bitter, too sour,
or too salty. However, humans seem to love sweetness
and most cannot get enough of eating sweet things. This
is probably what drove our ancestors to begin robbing
bee hives is prehistoric times. How early that might
have begun we don’t know, but paintings on the walls of
the Altamira Caves in Spain date to about 15,000 years
ago and show people on ladders robbing hives for the
honey. In historic times the Egyptians, Greeks, Romans,
and other early cultures all wrote about the importance
of honey as their main sweetener for foods and wine.
During medieval times dome-shaped beehive skeps were
in common use and the skep is still the most common
symbol for beekeeping.
In the New World, Native Americans in Mexico and
Central America developed bee keeping using a variety of
stingless bees. When early Spanish explorers conquered
those areas they reported the natives could get about two
kg of honey from one stingless bee colony, far less than
the bee colonies in Spain and Europe. The rst European
bees introduced into the New World are believed to have
arrived in Virginia in 1821. After the introduction of
European honey bees, some Indian tribes called them
“White Man’s ies” and referred to the newly introduced
white clover (Trifolium repens L.) that often accompanied
the spread of honey bees as “White Man’s foot” because
both clover and honey bees expanded with European
settlers. Reports from early New England colonists
reported that beekeeping was not pro table until 1851,
when Rev. Langstroth developed the removal frames in
hives that most people still use today.
By the mid-1800s European bees were fairly common
throughout the New World. In 1865 Hruschka invented
the centrifugal extractor, which greatly increased
commercial sales of honey because it could now be sold
as a liquid, as opposed to comb honey and the comb wax
could be turned into added pro t when made into candles
or other products.
Today, the United States is a major honey producer
but ranks far behind China and Turkey, but we have
made little effort to determine the contents of U.S.
domestic honey. Some work was done during the early
20th century but most of the research in the U.S. focused
on the chemical composition of honey and ways to identify
honey adulteration. Little effort during that time was
focused on the study of pollen in honey, even though
pollen composition was being recognized elsewhere as
the fastest and least expensive way to determine honey
oral types and geographical origins.
The history of the scienti c investigation of U.S. honey
and their pollen contents began in the early 1900s, when
Young, of the United States Department of Agriculture
(USDA), published a brief report on the analysis of
domestic honey produced in the U.S. He said goals were
to determine U.S. honey types, establish a variation range
for U.S. honey types, improve methods of U.S. honey
analysis, and see if pollen in honey could be used to
“judge the adulteration of the samples.” He reported that
“lower pollen counts” probably indicated altered honey.
After Young’s initial study in 1908, no other major
pollen study of domestic honey was conducted until the
early 1940s. Two USDA scientists, Todd and Vansell,
began their study of U.S. honey in 1940. They wanted
to determine how many pollen grains were present in
the nectar produced by different plants, how many
pollen grains were actually collected by honey bees from
various types of ower nectar, and how ef ciently could
bees remove various types of pollen from their honey
stomach during their return ight to the hive. Their
research represented years of effort and they examined
over 2,600 individual nectar samples. They caged bees
and fed them only solutions of clear syrup mixed with
pollen, or diluted honey they had analyzed. They also put
bees on blooming owers of different plants, then trapped
and dissected the bees’ honey stomach immediately after
they left the blooms.
Their research determined some dramatic differences
that nobody had realized and their results became the
basis for future research and studies that established
“coef cient values” for the expected pollen amounts found
in many types of honey. For example, they discovered
that the same amount of nectar from different plant
species contained different amounts of pollen, and that
bees could eliminate various amounts of pollen from
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42 November 2018
their honey stomachs before depositing it in the hive.
They found that bees full of nectar will rapidly lter out
certain types of pollen, but not other types from their
honey stomach using the action of the ventriculus and
their honey stopper. That process could eliminate much,
but not all, of the pollen the bee had collected with the
nectar. In one experiment they found that caged bees
fed a syrup solution containing 200,000 pollen grains/
cc of uid could eliminate most of the pollen if allowed
to y around for 15 minutes in a caged area with no
food. In other words, after collecting nectar, during a
normal return trip of 15 minutes to a hive, bees could
effectively remove, up to 90% of some pollen types from
their honey stomach. Other parts of the Todd and Vansell
study focused on determining the amount of pollen found
naturally in the nectar of many different plant species.
For example, they found that the nectar/pollen ratio for
reweed nectar contained only about 220 pollen grains/cc
of nectar, however, the nectar/pollen ratio for privet was
about 6,130 pollen grains/cc. They concluded that the
many different nectar/pollen ratios they had calculated
might help determine the true amounts of expected
pollen in honey. They found that depending on how long
bees took to return to the hive determined how much of
the pollen in the nectar they could eliminate. They also
discovered that the bigger the pollen grains the faster and
more ef cient the bees were at eliminating those types.
Therefore, large pollen types found in the owers and
nectar of magnolia and tulip trees could be eliminated
rapidly while smaller pollen grains in the owers of sweet
clover, blue weed and forget-me-nots could not.
After the Todd and Vansell report in the 1940s, there
were only a very few minor studies or mention of the
pollen contents in U.S. honey. It wasn’t until the 1970s,
and early 1980s, that the rst extensive pollen studies of
domestic U.S. honey were conducted by Meredith Lieux
at Louisiana State University. Her research represented
the rst U.S attempt to produce detailed pollen analyses
of honey, and she was the rst to use the combination of
pollen types as a guide to determining the geographical
location where each honey sample had been produced.
She was also the rst to use tracer spores to determine
the precise pollen concentration values (the amount of
expected pollen) in different types of Louisiana honey.
Since Lieux’s studies in the 1970s and early 1980s
there were only two additional studies of pollen in U.S.
honey, both during the last part of the 20th century
in the 1990s. The rst one was a study by Jonathan
White and Vaughn Bryant examining the chemical and
pollen properties of different mesquite and cat’s claw
acacia honey samples collected from regions in Texas
and Arizona. The focus of their research was to search
for ways to identify adulteration of honey using both
stable sugar isotope levels and pollen. They focused on
samples of mesquite and cat’s claw acacia honey that
were purported to be either adulterated or uni oral types;
however, their research showed otherwise. Their second
study focused on ways to identify uni oral orange blossom
honey based on both pollen percentages and levels of
Methyl Anthranilate.
The only published pollen study of U.S. honey this
century is the one by Gretchen Jones and Vaughn
Bryant on the honey from East Texas. The apparent
reason why in more than 100 years there have been so
few published studies of U.S. honey types is a re ection
of why so few people have the skills to analyze honey.
Learning to analyze the pollen trapped in honey requires
a broad understanding of botany and bee biology. Each
study is also very time-consuming and requires both
concentration and an inquisitive mind.
The process of recovering pollen from honey is not
complicated and it can be done fairly easily provided one
has the right kind of equipment and in some cases the right
type of laboratory. Because of the role pollen analyses play
in honey and honeybee research, it is essential that pollen
recovery techniques produce accurate and repeatable
results. Extracting pollen from honey is not a dif cult
process. The original recommended standard procedure
Citrus Mint Mimosa
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November 2018 BEE CULTURE 43
established during the early 1900s was to dilute 10 g
of honey with 20 ml of water, centrifuge the solution at
as slow speed of 2,500 revolutions per minute (RPM) for
ve minutes and then examine what was retained in the
bottom of the centrifuge tube. Subsequent researchers
claimed other techniques were better. Some of the later
techniques focused on using a quantity of honey ranging
from one – 20 g, while the amount of water used to dilute
the honey ranged from 20 – 100 ml; centrifugation speeds
also varied from 2,000 to 4,500 RPM and centrifugation
times varied from one minute to 10 minutes.
One of the reasons there were so many pollen recovery
techniques suggested is because often there really isn’t
much pollen in honey. The loss of any pollen from honey
can create problems in classifying the honey’s true nectar
sources and determining the honey’s geographic location.
Think of it this way, suppose you had a bag of 20 marbles
with 10 marbles that are black and 10 that are white.
What if you reach into the bag and take out a handful
and discard them? Now count what marbles you have
left. Do you still have 50% of both colors? I doubt it. In
other words, you no longer have a true representation of
what was originally in the bag of marbles. Honey is the
same way, if pollen is inadvertently discarded, you no
longer have a true pollen pro le of what was originally
in the honey.
The two most successful pollen extraction techniques,
proven through experimentation, is ltering honey or
diluting it in alcohol before processing. The lter process,
developed in 1983, by Lutier and Vaissiere, consists of
using honey diluted with ample amounts of water and
carefully ltering through a cellulose lter with openings
no larger than two to three microns. Using that technique,
they lost no pollen, but the process is very time-consuming
and lters can become clogged with large molecules of
sugar. The other technique, developed in 2001, by Jones
and Bryant, is to dilute 10 g of honey in 100 ml of ethyl
alcohol (ETOH) instead of water and then centrifuge the
diluted solution. By using ETOH the speci c gravity of the
honey/ETOH solution is lowered to 0.7 allowing all the
pollen to sink quickly when centrifuged. Their published
data showed it was as effective as the Lutier and Vaissiere
lter technique, but it was considerably easier and faster
to use. When water (speci c gravity of 1.0) is used to
dilute honey, without pressure ltering it, some pollen
can continue to oat after it has been centrifuged even
at high speeds for long periods of time, thus some pollen
can be inadvertently discarded.
Beekeepers are thrilled to nd out what their bees are
collecting to make honey. They like to use the information
gained from pollen analyses of their honey to increase sales
and also brag about their good honey. Some beekeepers
then decide that they can get results much faster if “they”
could do their own pollen study. Some beekeepers call me
and say they want to visit my lab for a few days to learn
how to do this. I try to discourage those eager callers
not because of my unwillingness to spend time with
them but because a few days is totally inadequate for
learning this complex type of analysis. I usually respond
to these enquires with a positive reply offering to work
with them if they wish, but adding that, “I have been
doing this for over 40 years and I am just now getting
good at it!” Being able to analyze the pollen contents of
honey samples requires a “long learning curve!” This is
not to say that it is impossible for a beekeeper to learn
how to do this, but most of them do not have the needed
scienti c background, the equipment to do the extraction
process, or the pollen reference collections needed to help
them identify the potential thousands of pollen types they
might nd in their samples.
To analyze pollen in honey there are many hurdles
one must overcome. Aside from the botanical and
entomological academic training that is needed, perhaps
the biggest hurdle is learning how to identify the pollen
types one might nd in honey samples and then know
the geographical regions where all the different plants
producing the pollen in the honey live. According to recent
botanical records, worldwide there are over 352,000
angiosperms ( owering plants) from which honey bees can
collect pollen and/or nectar. Of that total about 17,000
species are native to the United States. An estimated
additional 3,800+ ornamental owering plants have been
introduced into the U.S., not counting hundreds or maybe
thousands of additional species of introduced agricultural
plants. Each species of angiosperms produces a unique
pollen type that is different from all others. Many can
be fairly easy to identify using a light microscope at
magni cations up to 1,200. For other pollen species the
only way to be certain of the precise taxon is to use the
higher resolution ability of either or both scanning and
transmission electron microscopes to distinguish the
very nite differences that exist between types. The next
major hurtle in doing this type of research is making a
modern pollen reference collection from known plant
species and then using that collection for later comparison
with types of pollen found in honey samples. Some help
Composites. Clover.
There are over 352,000
angiosperms ( owering
plants) from which honey
bees can collect pollen
and/or nectar.
November2018.indd 43 11/14/2018 2:31:47 PM
44 November 2018
can also come from pollen web sites on the internet and
from published articles and pollen atlases. Nevertheless,
searching for the identity of some pollen types in honey
can take hours or days and in the end it might remain
unknown. Perhaps the nal hurdle is recognizing where
a honey originated. Many consumers want to buy “local
honey” from the area where they live, however, all too often
honey labeled as being “local” is not local at all. How do
we know this? By examining the pollen in the honey and
realizing that the nectar sources they represent do not
come from plants living in the “local area!”
If pollen studies of honey require so much training
and such a long learning curve, which most students
these days are not willing to do, then why not use a
different way to test honey, such as isotopes? For years
we have known that various types of adulteration can be
detected by using carbon stable isotopic ratio analysis
(SIRA). Variations from established isotope ratios in
honey often suggest some degree of adulteration created
by the addition of high fructose corn syrup or cane syrup.
However, sugar added to honey made from beets or rice,
is not often detectable using only stable isotope testing.
Other isotopes in the proteins of pollen grains in
honey include variations of carbon, hydrogen, nitrogen,
and oxygen that can be used to reveal the general climate
and environment where a honey sample is produced, and
in some cases can be used to identify types of pollen.
However, none of the individual isotope values found
in honey proteins or pollen is usually unique enough to
identify a precise geographical region or the percentage
of some pollen type in a sample. Sometimes, the use of
multivariate statistical analysis of the various isotope
signatures can help to discriminate between different
geographical locations.
What about the DNA of honey? Why not analyze
the deoxyribonucleic acid (DNA) properties of honey?
Recently, there has been signi cant progress in this
area with the development of pyrosequencing and the
use of barcoding strands of DNA. For plants, the ideal is
to barcode selective DNA strands that have one or a few
standard loci that can be sequenced easily and reliably
in large sample sets. Comparisons of those sequenced
data against available plant DNA data bases enable
speci c pollen types to be distinguished from one another.
DNA barcoding can also be used to link speci c pollen
types in honey bee pollen pellet studies. The screening
process is becoming faster and less expensive than having
palynologists spend hours or days sorting through pellets
to identify the many pollen types and the ratios of each
type. However, there are still many challenges, as noted by
Richardson and his research group in 2017. When using
pollen pellets in tests that paired DNA data against pollen
data identi ed by skilled palynologists, Richardson’ group
found that neither technique was perfect. Nevertheless,
the eld of DNA analysis of honey and pollen pellets is
evolving rapidly with new studies in genetics using gene-
based results. New testing comparing DNA data against
actual pollen counts are showing great promise and
helping to re ne the gene-based techniques. It appears
that for large pollen pellet studies funded by state or
federal agricultural agencies, emerging genetic techniques
might prove to be the fastest and most ef cient way to
Honey samples at nish of processing.
study vast amounts of data collected from large areas.
Currently, a major problem is that not everyone has
access to the equipment or expertise needed to conduct
isotope or gene-based analysis of either pollen pellets
or honey. Therefore, there is still room for palynologists
using light microscopy to analyze both honey and pollen
pellets. Small studies that focus on limited numbers of
samples, which are often requested by local beekeepers,
may not warrant the use of expensive methods for testing.
Pollen studies conducted by palynologists using the limits
of light microscopy are still adequate for many types of
honey and pollen pellet studies where quick results are
needed and where the main concern might be determining
the primary (but not all) oral types and not needing an
ID down to the species level.
In recent years, there have been advancements
in using three-dimensional synchronous uorescence
spectroscopy (3-D SFS) to provide unique “ ngerprint”
types of identi cation for the phenolic contents in honey.
Those identi cations provide good signatures for the
oral sources and can identify the geographic origin of
honey samples. Nevertheless, melissopalynology studies
of honey using standard light microscopy to determine
the geographical origin of speci c honey samples (local
vs. not local) is still less expensive and faster than using
other techniques.
On the fringes of current honey research there are
researchers searching for ways to use liquid and gas
chromatography to identify the amino acids in honey and
thus identify the various honey types. Different amino
acid patterns exist in different types of honey and if you
have a good database of the expected amino acids in
honey, then by applying discriminant analysis one can
distinguish some key honey types.
Volatiles contribute signi cantly to the avor of
honey and variations in taste result from different nectar
types. The isolation and analysis of phenolic acids and
the volatile avonoid components in honey is dif cult,
but possible. Previous attempts con rm that a careful
analysis of the volatiles in honey could become a useful
tool for determining nectar sources. However, the volatile
components could be changed and altered depending
on how the honey was treated and stored before testing
it. High performance liquid chromatography has been
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November 2018 BEE CULTURE 45
Honey samples.
successfully used to characterize the avonoid patterns
in speci c types of honey, such as citrus, sun ower,
lavender, rosemary, and heather. Some believe that
avonoid patterns could become a good way to determine
the oral contents and geographical origins of honey,
provided one could develop a good database and use
multivariate statistics.
The mineral and trace elements in honey samples
have shown some promise when used to indicate types
of environmental pollution that can pinpoint certain
geographical locations as the origin of honey samples.
Tests of the composition of organic acids in honey
have also demonstrated that it might become useful in
identifying speci c uni oral types of honey.
Unfortunately, all of these other methods of honey
and pollen pellet identi cation thus far suffer from some
type of problem. For some, the problems focus on the
need for special and expensive laboratory equipment.
Others are too time-consuming and require an extensive
database before the results can be determined. Some
methods are too costly to use routinely; others require
skilled technicians or analysts. It seems there is not yet
an inexpensive, foolproof, or simple way to verify the oral
and/or geographical origin of a honey sample.
In summary, melissopalynology still has a promising
future as a way to identify the oral sources, potentials of
adulteration, possibility of blending, and the determination
of local or foreign origin of a sample. Worldwide
melissopalynology currently remains the least expensive
and quickest method to get reliable answers, provided the
pollen extraction and analysis is conducted by competent
individuals. Nevertheless, the rapid advancements in
the eld of gene-based methods of pollen identi cation
may someday make melissopalynology, conducted by
individuals using a light microscope, obsolete!
Vaughn M. Bryant, PhD, Professor and Director, Palynology
Laboratory, Department of Anthropology, Texas A&M University,
College Station. www.//
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