Original article
Microscopic detection of adulteration of honey
with cane sugar and cane sugar products
JD Kerkvliet M Shrestha K Tuladhar H Manandhar
1
Regional Inspectorate
for Health Protection Food Inspection Service, Hoogte Kadijk 401, 1018 BK Amsterdam, The Netherlands;2
Beekeeping Training
and ExtensionSupport Project (BETRESP),
c/o HMG/SNV
Nepal,
PO Box 1966 Kathmandu,Nepal
(Received
6 October 1994;accepted
30January 1995)
Summary —
Amicroscopic procedure
is described to detect adulteration of honey with cane sugar,acid-hydrolized
cane sugar syrup or with’honey’
obtained fromfeeding
sugar to bees. The method consists ofpreparing
a microscope slide of thehoney sample
andtaking
up the sediment inglycerin jelly
in the same way as in classical pollenanalysis. Microscopic analysis
ispreferably
done
by polarization microscopy using
crossed polars and a first-order red retardation plate.Adulteration of
honey
with white or brown cane sugar and syrups derived from cane sugar is shownby
the presence of manyparenchyma
and sclereid cells,single rings
fromring
vessels andepidermis cells. These cells are very characteristic and
originate
from the sugar cane stem. Even alow percentage of cane sugar
(products)
may be detected in this way.Analysis
of 10 selectedsamples
ofhighly
adulteratedhoney
from thePhilippines
andNepal
is described.Upon
furtherstudy
it turned out that no false-positive orfalse-negative
results were obtained.Sugar
cane honeysdid not contain sugar cane
plant
cells and so no false positives were observed, even in sugar canehoney.
honey
/ adulteration / cane sugar/ microscopy
INTRODUCTION
Adulteration of
honey
is a well-knownprob-
lem and many methods of
analysis
are avail-able to detect falsification with various
types
of sugars and with
inexpensive
sugar syrups.In some
tropical countries, honey
for saleon the local market may be adulterated
by directly adding crystallized
cane sugar, canesugar syrup, invert sugar syrup obtained
by heating slightly
acidified cane sugar, or’honey’ obtained
fromfeeding
sugar to bees.By carrying
out the usual chemical determi-nations,
such as theglucose, fructose,
sucrose and
hydroxymethylfurfural (HMF)
content and the diastase
index,
these adul- terations ofhoney
caneasily
be detected(Codex, 1989; White, 1979).
However,
in manydeveloping
countriesa
laboratory
for the above-mentioned routineanalyses
is notalways
available.However, by simple microscopic analysis
it ispossible
to detect adulteration of
honey
with canesugar and
products
derived from canesugar. Cane sugar has a built-in indicator of
origin;
it contains many characteristic par-ticles, originating
from the sugar cane stem:parenchyma,
sclereid andepidermis cells, single rings
ofring
vessels and sugar cane starch(Kerkvliet, 1982).
Theseparticles
arepresent
in raw(brown)
cane sugar, refined white cane sugar and dark brown(B-type)
molasses cane sugars from all over the
world,
but not in beet sugar,maple
sugar and Indonesianpalmtree
sugar.In the literature no
specific
method forthe
microscopic analysis
ofhoney
of canesugar
particles
is described. Some authors mention the presence of other structuredparticles
besidespollen
in thehoney
sedi- ment, but detailed identification ofvegetable
cells is not
given (eg,
Evenius andFocke, 1967;
Sanchoet al, 1991).
By centrifuging
a 1:2honey/water
solu-tion,
as used in classicalpollen analysis,
in cases where cane sugar and cane sugar
products
are used foradulteration,
the residue obtained contains the characteris- tic sugar caneplant
cells in addition topollen, honeydew
elements and oxalatecrystals.
Aftermounting
the water washedresidue in
glycerin jelly, microscopic
anal-ysis
is carried out.Using
crossedpolars
and a first-order red retardation
plate,
thepresence of cane sugar is
immediately
revealed
by
abright lightening
of the char- acteristic sugar caneplant
cells.Typical
forms are
pictured
infigures
1-7. The ace-tolysis
method(Louveaux et al, 1978),
awell-known
procedure
inmelissopalynol-
ogy in which the
pollen
sediment is acetol-ysed by
aceticanhydride/sulphuric
acidmixture to obtain clear
pollen
cellwalls,
cannot be used for detection of these
plant
cells because of destruction of the cells
during
theprocedure.
Polarization
microscopy
isespecially
use-ful for this
type
ofanalysis.
Between crossednicols, vegetable
cells with cell walls con-sisting mainly
of cellulose show first-order white interference colours on a black back-ground. Many crystals
and native starchgrains
are also visible. Howeverpollen, yeasts
andfungal
spores are notoptically
active and hence cannot be seen between crossed nicols.
By inserting
a first-order red retardationplate (red I plate),
the black back-ground changes
to to wine-red.Pollen, yeasts
andfungal
spores can be distin-guished
but are not lit up. However theopti- cally
activeplant
cells(and crystals
andnative
starch)
dogive
second-orderbright
blue
(red I plus
whiteI)
or first-orderyellow (red
I minus whiteI)
interferencecolours, depending
on their orientation in the micro-scopic
field(Czaja, 1971).
In this
study
the results of themicroscopic analyses
for sugar canefragments
are com-pared
with theglucose,
fructose and sucrose content of thehoney
and with the HMF val-ues. In some
(sub)tropical
countries beesforage
on sapexuding
from cut or burntsugar cane
(Saccharum officinarum)
stems,therefore, microscopic analysis
of some sugar canehoneys
was also included in thisstudy.
MATERIALS AND METHODS
Samples
Honey samples
were obtained from thePhilip- pines during
a survey carried out in 1987, in thecourse of a
development project.
Twohighly
sus-pected samples, bought
on the local market, wereanalysed
at the FoodInspection
Service(Haar- lem),
The Netherlands.The other 8
honey samples
in thisstudy orig-
inated from
Nepal (Kathmandu, Terai). They
wereencountered
during
chemical andmicroscopic
routine
analyses
at the laboratories of BETRESP,a
joint development
project set upby
HisMajesty’s
Government ofNepal
and The Nether- landsDevelopment Organization
with the aimsof
training
beekeepers andimproving honey yield
and
honey quality. By analyzing
more than 300honey samples,
BETRESP has laid the basis forestablishing
a nationalhoney
standard inNepal.
Because some of the
samples
contained sugarcane
fragments they
were alsoanalyzed
at theRegional Inspectorate
for Health Protection FoodInspection
Service laboratories, Amsterdam, TheNetherlands.
Cane sugar samples were
bought
in localshops
in Kathmandu,Nepal.
Pure sugar canehoney samples
were obtained from thehoney
collection at the Food
Inspection
Service, Ams- terdam, The Netherlands. One sampleoriginated
from Cuba, the other from Madeira.
Methods
of analysis
Microscopic analyses
were carried out accord-ing
to the methodspublished by
the International Commission for Bee Botany(Louveaux
et al,1978) by dissolving
10 ghoney (or
canesugar)
in20 ml water,
centrifuging, washing
out the residue with water,centrifuging again
andtaking
up the residue in 100 μl water. For quantitativeanaly-
sis, a 10 μl suspension was placed on 1 cm2of the microscopic slide, dried and taken up inglyc-
erin
jelly.
Cane sugarparticles
were countedby using
crossedpolars
and a first-order red retar- dationplate
at amagnification
of 400 x. Theremaining
part of the suspension was placed ona second slide, dried and also taken up in
glycerin jelly.
This slide was used forqualitative
identifi-cation and, if necessary, for low counts in quan-
titative work
using
the samemicroscopic
tech- nique.Parenchyma
cells of cane sugar areroughly rectangular
with various dimensions; thelength
ofa cell is
mostly
50-100 μm, the widthapproxi- mately
40-60 μm. More round forms are alsopresent. Sclereid cells
(stone cells)
are rectan-gular, length approximately
100-200 μm, width 40-50 μm. Both type of cells possessoptically
active cell walls with blue
(orientation
north-east/south-west)
andyellow (orientation
north-west/south-east) polarization
colours. The sur-face of the
parenchyma
and the sclereid cells is characterizedby
many smallpits
with a diameterof about 2-3 μm. Each
pit
has apolarization
crossor a
single
beam withoptically
activesurroundings (yellow
andblue) (figs 1-3).
Because
they
are characterizedby
theirpits,
the number of
parenchyma
and sclereid cells were countedtogether
forquantitative
work andexpressed
as the total number in 10 ghoney.
Clusters and parts of cells were each counted as
one cell.
Single rings
ofring
vessels of cane sugar are also very characteristic. Their diameter is about 30-50 μm, and the thickness of therings
is 4-5 μm
(fig 4). They
show blue andyellow polar-
ization colours in a
single ring;
the colours areinterchanged by
small red(fig
5: black) bands.Single rings
were counted andexpressed
as thetotal number in 10 g
honey.
Epidermis cells of cane sugar are character- ized
by
theirundulatory long
cell walls; dimen-sions of one cell are about 15 μm
by
more than150 μm. The
long
cell walls showbright
bluepolarization
colours in the orientation north- east/south-west. The colour between the blue cell walls isbright yellow.
In the other direction(north-west/south-east)
thepolarization
coloursare reversed
(figs
6 and7). Epidermis
cells aremostly
present in thehoney
residue as clusters.Single
cells and clusters are counted andexpressed
as the total number in 10 ghoney.
HMF was determined
by
thehigh
pressureliquid chromatography (HPLC) technique (Jeuring, 1980);
sugar determinations(glucose,
fructoseand sucrose
content)
were also carried outby
anHPLC method.
pH
and electricalconductivity
weremeasured in a 20%
(m/m) honey (or
canesugar)
solution in distilled water
(Vorwohl, 1964),
theelectrical
conductivity
isexpressed
inμSiemens/cm
on thehoney
as such(not
ondry matter).
Water content was determinedby
therefractometer method.
RESULTS
The results of the chemical and
microscopic analyses
of 10 adulteratedhoney samples
from the
Philippines
andNepal
are shown intables I and II. These tables also
give
theresults of the
analyses
of atypical crystal-
lized normal
quality Nepalese
cane sugaralong
with 2types
offinely powdered
minorquality
molasses cane sugars.The sugar cane
honey samples
weredark-brown or black with a taste reminiscent of cane sugar.
Upon microscopic analysis
no sugar cane
plant
cells were observed.The total number of
pollen grains
insample
R 128 had a normal
value;
mostpollen
werefrom a
(wind-pollinated?)
Palmeaespecies.
Sample
A 485 did not containpollen,
but didhave lots of calcium oxalate
crystals.
DISCUSSION
Honey samples
From the chemical data of all 10
honey
sam-ples,
it is evident thatthey
arewholly
orpartly
adulterated or at leaststrongly
heated.From the
microscopic
data it follows that thesamples
are adulterated. There is nodirect correlation between the number of
cane sugar
fragments
and sucrose content, but all 8Nepalese samples
do have ahigh
number of cane sugar
fragments
and alarge
amount of
HMF,
an indication thatthey
areadulterated with
acid-hydrolyzed
cane sugar syrup. This does not hold for the 2Philip- pine honeys,
which containpractically
noHMF
(and they
have very low diastase num-bers,
not inserted in thetable)
and areprob- ably
adulteratedby mixing honey directly
with cane sugar.
The presence of many wheat starch
grains along
with sugar canefragments
insome of the
honey samples
may indicatethat
they
are adulterated with minorquality
molasse cane sugar syrup.
Microscopic analyses
The use of
polarization microscopy (crossed
nicols with first-order red retardation
plate)
makes the identification of cane sugar
frag-
ments very easy. In this way,
using
a totalmagnification
of 100 x a whole slide can bescreened for
parenchyma
and sclereidcells, single rings
andepidermis
cells.Especially,
the
rings
can be seen at the firstglance.
For proper identification of
epidermis
andespecially parenchyma
and sclereidcells,
it is better to use ahigher magnification (400 x)
with the samemicroscopic technique
orwith crossed nicols
only.
In this way the characteristicpits
in theparenchyma
cellwalls can
easily
be detected.The mean value of
parenchyma
and scle-reid cells in this 10 adulterated
honey
sam-ples together
with the standard deviation and the range is shown in table III.The number of
parenchyma
and sclereid cells in atypical
white cane sugarsample
fromNepal
is 5 902 in 10 g. In an earlierstudy (Kerkvliet, 1982),
it was found that in varioustypes
of cane sugaroriginating
fromdifferent
countries,
the number ofsingle rings ranged
between 76 and 595 in 10 g and the number ofepidermis
cells between 25 and 222 in 10 g.Parenchyma
and scle-reid cells are
present
in a fargreater
num-ber, usually
between 3 000 and 6 000 in 10 g. It was also found that cane sugars con- tainedparenchyma,
sclereid andepidermis
cells and
rings.
From this observation itseems
highly unlikely
thatfalse-negative
results are obtained in
screening honey samples
for sugar canefragments.
False
positive
results arehighly unlikely.
In the
honey samples
frombeekeepers
whoare under
supervision
of theNepalese
BETRESP
project,
no sugar canefragments
were found upon
microscopic analysis.
Chemical
analyses
also showed normal val-ues.
To make an estimation of the limit of detection of the presence of cane sugar in
honey,
a minimum of about 30parenchyma
and sclereid cells in 10 g of
honey
can eas-ily
be detectedby screening
the 1cm 2
areaof the
microscopic
slide of thesample.
Fromthis data and
taking
into account the above-mentioned natural variation in the number of
parenchyma
and sclereid cells in canesugar, it follows that even 1 or 2
percent
cane sugar can be detected in a
simple
wayby
this method.CONCLUSIONS
Microscopic screening
ofhoney samples
forsugar cane
fragments
is an additionalmethod to detect even a minor addition to
honey
of cane sugar, inverted cane sugar syrup and’honey’
from cane sugar fed to bees. As theinvestigated
sugar cane(S
offic-inarum) honey samples
did not contain theplant
cells from sugar cane described in thisstudy
we may conclude that the presence of sugar canefragments
shows that thehoney sample
is adulterated.By counting
the sugar cane
fragments
an indication of the amount of cane sugar in thehoney
sam-ple
can be obtained. The method is espe-cially
usuful in theanalysis
ofhoney
fromdeveloping
countries and can be done withsimple equipment.
In combination with the HMF value themicroscopic
method may dif- ferentiate betweenheating
and adulteration.Résumé —
Diagnostic
enmicroscopie
de la falsification du miel par du sucre de
canne et des
produits
à base de sucre decanne. On décrit une méthode microsco-
pique
pour détecter les falsifications de mielavec du sucre de canne, du
sirop
de sucre decanne et du «miel» de sucre. La méthode a
été mise au
point
pour des miels locaux pro- venant derégions (sub)tropicales.
Le sucrede canne
raffiné,
le sucre de canne brut et lessirops
de sucre invertipréparés
avec dusucre de canne renferment
toujours
ungrand
nombre de cellules
parenchymateuses
destiges
de canne à sucre, des cellules sclé- reuses, des anneaux isolés des vaisseaux annelés et des cellules del’épiderme.
Cesparticules
sont absentes du sucre de bette- rave, du sucre d’érable et du sucre depalme.
On ne les a pas trouvées non
plus
dans les2 échantillons
analysés
de miel de canne àsucre
(Saccharum officinarum) (tableau II).
Ces cellules
végétales caractéristiques
ontété mises en évidence sur une
préparation microscopique
de miel réalisée selon laméthode
classique (Louveaux et al, 1978).
Le résidu est
repris
dans 100μl
d’eau. 10μl
de ce volume sont étalés sur une
lame,
lereste sur une seconde lame et les 2 lames sont séchées. Les résidus sont montés dans de la
gélatine glycérinée
de Kaiser et étu-diés au
microscope.
L’étude a été faite enpartie
aumicroscope polarisant
avec uneplaque
rouge I. Des cellulesparenchyma-
teuses,scléreuses,
les anneaux et les cel- lules del’épiderme
ont été identifiés. Uneanalyse quantitative
estégalement possible
sur une
préparation
de 1cm 2 .
Les cellulesparenchymateuses,
de formegénéralement rectangulaire
ouronde,
mesurent 50-100μm de
long
et 40-60 μm delarge.
Les cel-lules
scléreuses, rectangulaires,
mesurentenviron 100-200 μm de
long
et 40-50 μm delarge.
Les 2types
de cellulespossèdent
des
parois optiquement
actives avec les cou-leurs de
polarisation
bleue(orientation
nord-est/
sud-ouest)
etjaune (orientation
nord-ouest/sud-est).
La surface des cellules estgarnie
de nombreusesponctuations (2-
3 μm de
diamètre), chaque ponctuation
pos- sédant une croix depolarisation
ou unebande avec un
entourage optiquement
actif(jaune
etbleu) (figs 1-3).
Les anneaux desvaisseaux annelés ont 30-50 μm de dia-
mètre,
leursparois
uneépaisseur
de 4-5 μm. Les anneaux
présentent
des couleurs depolarisation
bleue etjaune
avec d’étroitesbandes rouges
(figs 4-5).
Les cellules del’épiderme,
±150 x 15 μm,possèdent
delongues parois
ondulées etprésentent
descouleurs de
polarisation
bleu clair etjaune (figs 6-7).
Dans les 10 miels falsifiésanaly- sés, provenant
desPhilippines
et duNépal,
on a trouvé entre 455 et 7 845 cellules paren-
chymateuses
etscléreuses,
de 8 à 1 025anneaux et de 2 à 512 cellules de
l’épiderme
dans 10 g de miel
(tableau II).
Cette méthodepermet
de détecter des falsifications de miels locaux avec seulement 1 à 2% de sucre decanne
(ou
deproduits
à base de sucre decanne).
miel / falsification / sucre de canne /
microscopie
Zusammenfassung — Mikroskopische
Methode zum Nachweis von Verfäl-
schungen
vonHonig
mit Rohrzucker undRohrzuckerprodukten.
Es wird über einemikroskopische
Methode zum Nachweisvon
Verfälschungen
desHonigs
mit Rohr-zucker, Rohrzuckersirup
oder Rohrzucker-fütterungshonig
berichtet. Die Methodewurde zur
Untersuchung
von lokalen Honi- gen aus(sub)tropischen
Gebieten ent-wickelt. Weisser und brauner Rohrzucker sowie aus Rohrzucker bereitete
(Invert)Zuckersirupe
enthalten immer eine grosse AnzahlParenchymzellen
desZuckerrohrstengels, Sklereidzellen,
einzelneRinge
vonRinggefässen
undEpidermis-
zellen.
Rübenzucker,
Ahornzucker und Palmzucker besitzenderartige
Bestandteilenicht;
auch in zwei Proben von Zuckerrohr-honigen (Saccharum officinarum)
wurde kei-nes dieser
Fragmente gefunden.
Zum Nach-weis dieser charakteristischen
pflanzlichen
Zellen wird ein
mikroskopisches Präparat
des
Honigs
nach der klassischen Methodehergestellt (auflösen
von 10 gHonig
in 20 mlWasser, zentrifugieren,
Rückstand mit Was-ser
auswaschen, zentrifugieren).
Der aus-gewaschener
Rückstand wird in 100μl
Was-ser
aufgenommen,
10μl
werden auf 1 cm2 desObjektglasses aufgetragen
undgetrock-
net; dasübrige
Volumen wird auf ein zwei- tesObjektglas aufgetragen
und ebenfallsgetrocknet.
Beide Rückstände werden zurmikroskopischer Untersuchung
inGlycerin- gelatine
nach Kaiseraufgenommen.
ZurUntersuchung
erwies sich die Polarisati-onsmikroskopie
mit der Rot I Platte als vor-teilhaft. Identifiziert werden
Parenchymzel- len+Sklereidzellen, Ringe
undEpidermiszellen;
auch einequantitative
Aus-zählung
auf dem 1 cm2Präparat
istmög-
lich.
Parenchymzellen
sind meistensrechwinklig
oderrund,
50-100 μmlang
und40-60 μm breit. Sklereidzellen sind recht-
winklig, ungefähr
100-200 μmlang
und40-50 μm breit. Beide
Zelltypen
besitzenoptisch
aktive Zellwände mit blauen(Lage nordost/südwest)
undgelben (Lage
nord-west/südost)
Polarisationsfarben. Die Ober- fläche der Zellen hat vieleTüpfel (2-3
μmDurchmesser)
die ein Polarisationskreuz oder einen Balken mitoptisch
aktiverUmge- bung (gelb
undblau)
aufweisen(Abb 1-3).
Einzelne
Ringe
vonRinggefässen
habeneinen Durchmesser von 30-50 μm, die Dicke der
Ringwände beträgt
4-5 μm. DieRinge
zeigen
blaue undgelbe
Polarisationsfarben mit schmalen roten Balken(Abb 4,5). Epi-
dermiszellen haben
Abmessungen
von±150 x ±15
μm
und besitzengewölbte lange
Zellwände. Sie
zeigen
helle blaue undgelbe
Polarisationsfarben
(Abb 6,7).
In 10 Probenvon
(teilweise)
verfälschtenHonigen
vonden
Philippinen
und ausNepal
wurden 455bis 7845
Parenchymzellen
+ Sklereidzel-len,
8 bis 1025Ringe
und 2 bis 512Epi-
dermiszellen pro 10 g
Honig gefunden.
Ver-fälschungen
von lokaleHonigen
mit nur 1oder 2 Prozenten
Rohrzucker(produkten)
sind mit dieser Methode nachweisbar.
Mikroskopie
/Honig
/ Rohrzucker / Ver-fälschung
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