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Microscopic detection of adulteration of honey with canesugar and cane sugar products

Jd Kerkvliet, M Shrestha, K Tuladhar, H Manandhar

To cite this version:Jd Kerkvliet, M Shrestha, K Tuladhar, H Manandhar. Microscopic detection of adulteration of honeywith cane sugar and cane sugar products. Apidologie, Springer Verlag, 1995, 26 (2), pp.131-139.<hal-00891253>

Original article

Microscopic detection of adulteration of honeywith 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 Extension Support Project (BETRESP),c/o HMG/SNV Nepal, PO Box 1966 Kathmandu, Nepal

(Received 6 October 1994; accepted 30 January 1995)

Summary &mdash; A microscopic procedure is described to detect adulteration of honey with cane sugar,acid-hydrolized cane sugar syrup or with ’honey’ obtained from feeding sugar to bees. The methodconsists of preparing a microscope slide of the honey sample and taking up the sediment inglycerin jelly in the same way as in classical pollen analysis. Microscopic analysis is preferablydone 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 many parenchyma and sclereid cells, single rings from ring 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 of highly adulterated honey from the Philippines and Nepal is described. Upon furtherstudy it turned out that no false-positive or false-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-known prob-lem and many methods of analysis are avail-able to detect falsification with various typesof sugars and with inexpensive sugar syrups.In some tropical countries, honey for saleon the local market may be adulterated bydirectly adding crystallized cane sugar, cane

sugar syrup, invert sugar syrup obtained byheating slightly acidified cane sugar, or’honey’ obtained from feeding sugar to bees.By carrying out the usual chemical determi-nations, such as the glucose, fructose,sucrose and hydroxymethylfurfural (HMF)content and the diastase index, these adul-terations of honey can easily be detected(Codex, 1989; White, 1979).

However, in many developing countriesa laboratory for the above-mentioned routineanalyses is not always available. However,by simple microscopic analysis it is possibleto detect adulteration of honey with canesugar and products derived from canesugar. Cane sugar has a built-in indicatorof origin; it contains many characteristic par-ticles, originating from the sugar cane stem:parenchyma, sclereid and epidermis cells,single rings of ring vessels and sugar canestarch (Kerkvliet, 1982). These particles arepresent in raw (brown) cane sugar, refinedwhite cane sugar and dark brown (B-type)molasses cane sugars from all over the

world, but not in beet sugar, maple sugarand Indonesian palmtree sugar.

In the literature no specific method forthe microscopic analysis of honey of canesugar particles is described. Some authorsmention the presence of other structured

particles besides pollen in the honey sedi-ment, but detailed identification of vegetablecells is not given (eg, Evenius and Focke,1967; Sancho et al, 1991).

By centrifuging a 1:2 honey/water solu-tion, as used in classical pollen analysis,in cases where cane sugar and cane sugarproducts are used for adulteration, theresidue obtained contains the characteris-tic sugar cane plant cells in addition topollen, honeydew elements and oxalatecrystals. After mounting the water washedresidue in glycerin jelly, microscopic anal-ysis is carried out. Using crossed polarsand a first-order red retardation plate, thepresence of cane sugar is immediatelyrevealed by a bright lightening of the char-acteristic sugar cane plant cells. Typical

forms are pictured in figures 1-7. The ace-tolysis method (Louveaux et al, 1978), awell-known procedure in melissopalynol-ogy in which the pollen sediment is acetol-ysed by acetic anhydride/sulphuric acidmixture to obtain clear pollen cell walls,cannot be used for detection of these plantcells because of destruction of the cells

during the procedure.Polarization microscopy is especially use-

ful for this type of analysis. Between crossednicols, vegetable cells with cell walls con-sisting mainly of cellulose show first-orderwhite interference colours on a black back-

ground. Many crystals and native starchgrains are also visible. However pollen,yeasts and fungal spores are not opticallyactive and hence cannot be seen betweencrossed nicols. By inserting a first-order redretardation plate (red I plate), the black back-ground changes to to wine-red. Pollen,yeasts and fungal spores can be distin-guished but are not lit up. However the opti-cally active plant cells (and crystals andnative starch) do give second-order brightblue (red I plus white I) or first-order yellow(red I minus white I) interference colours,depending on their orientation in the micro-scopic field (Czaja, 1971).

In this study the results of the microscopicanalyses for sugar cane fragments are com-pared with the glucose, fructose and sucrosecontent of the honey and with the HMF val-ues. In some (sub)tropical countries beesforage on sap exuding from cut or burntsugar cane (Saccharum officinarum) stems,therefore, microscopic analysis of somesugar cane honeys was also included in thisstudy.

MATERIALS AND METHODS

Samples

Honey samples were obtained from the Philip-pines during a survey carried out in 1987, in thecourse of a development project. Two highly sus-pected samples, bought on the local market, wereanalysed at the Food Inspection Service (Haar-lem), The Netherlands.

The other 8 honey samples in this study orig-inated from Nepal (Kathmandu, Terai). They wereencountered during chemical and microscopicroutine analyses at the laboratories of BETRESP,a joint development project set up by HisMajesty’s Government of Nepal and The Nether-lands Development Organization with the aimsof training beekeepers and improving honey yieldand honey quality. By analyzing more than 300honey samples, BETRESP has laid the basis forestablishing a national honey standard in Nepal.Because some of the samples contained sugarcane fragments they were also analyzed 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 the honeycollection at the Food Inspection Service, Ams-terdam, The Netherlands. One sample originatedfrom Cuba, the other from Madeira.

Methods of analysis

Microscopic analyses were carried out accord-ing to the methods published by the InternationalCommission for Bee Botany (Louveaux et al,1978) by dissolving 10 g honey (or cane sugar) in20 ml water, centrifuging, washing out the residuewith water, centrifuging again and taking up theresidue in 100 &mu;l water. For quantitative analy-sis, a 10 &mu;l suspension was placed on 1 cm2 ofthe microscopic slide, dried and taken up in glyc-erin jelly. Cane sugar particles were counted byusing crossed polars and a first-order red retar-dation plate at a magnification of 400 x. Theremaining part of the suspension was placed ona second slide, dried and also taken up in glycerinjelly. This slide was used for qualitative identifi-cation and, if necessary, for low counts in quan-

titative work using the same microscopic tech-nique.

Parenchyma cells of cane sugar are roughlyrectangular with various dimensions; the length ofa cell is mostly 50-100 &mu;m, the width approxi-mately 40-60 &mu;m. More round forms are alsopresent. Sclereid cells (stone cells) are rectan-gular, length approximately 100-200 &mu;m, width40-50 &mu;m. Both type of cells possess opticallyactive cell walls with blue (orientation north-east/south-west) and yellow (orientation north-west/south-east) polarization colours. The sur-face of the parenchyma and the sclereid cells ischaracterized by many small pits with a diameterof about 2-3 &mu;m. Each pit has a polarization crossor a single beam with optically active surroundings(yellow and blue) (figs 1-3).

Because they are characterized by their pits,the number of parenchyma and sclereid cellswere counted together for quantitative work andexpressed as the total number in 10 g honey.Clusters and parts of cells were each counted asone cell.

Single rings of ring vessels of cane sugar arealso very characteristic. Their diameter is about30-50 &mu;m, and the thickness of the rings is 4-5 &mu;m (fig 4). They show blue and yellow polar-ization colours in a single ring; the colours areinterchanged by small red (fig 5: black) bands.Single rings were counted and expressed as thetotal number in 10 g honey.

Epidermis cells of cane sugar are character-ized by their undulatory long cell walls; dimen-sions of one cell are about 15 &mu;m by more than150 &mu;m. The long cell walls show bright bluepolarization colours in the orientation north-east/south-west. The colour between the bluecell walls is bright yellow. In the other direction(north-west/south-east) the polarization coloursare reversed (figs 6 and 7). Epidermis cells aremostly present in the honey residue as clusters.Single cells and clusters are counted andexpressed as the total number in 10 g honey.

HMF was determined by the high pressureliquid chromatography (HPLC) technique (Jeuring,1980); sugar determinations (glucose, fructoseand sucrose content) were also carried out by anHPLC method. pH and electrical conductivity weremeasured in a 20% (m/m) honey (or cane sugar)solution in distilled water (Vorwohl, 1964), theelectrical conductivity is expressed in

&mu;Siemens/cm on the honey as such (not on drymatter). Water content was determined by therefractometer method.

RESULTS

The results of the chemical and microscopicanalyses of 10 adulterated honey samplesfrom the Philippines and Nepal are shown intables I and II. These tables also give theresults of the analyses of a typical crystal-lized normal quality Nepalese cane sugaralong with 2 types of finely powdered minorquality molasses cane sugars.

The sugar cane honey samples weredark-brown or black with a taste reminiscentof cane sugar. Upon microscopic analysisno sugar cane plant cells were observed.The total number of pollen grains in sampleR 128 had a normal value; most pollen werefrom a (wind-pollinated?) Palmeae species.

Sample A 485 did not contain pollen, but didhave lots of calcium oxalate crystals.

DISCUSSION

Honey samples

From the chemical data of all 10 honey sam-ples, it is evident that they are wholly orpartly adulterated or at least strongly heated.From the microscopic data it follows thatthe samples are adulterated. There is nodirect correlation between the number ofcane sugar fragments and sucrose content,but all 8 Nepalese samples do have a highnumber of cane sugar fragments and a large

amount of HMF, an indication that they areadulterated with acid-hydrolyzed cane sugarsyrup. This does not hold for the 2 Philip-pine honeys, which contain practically noHMF (and they have very low diastase num-bers, not inserted in the table) and are prob-ably adulterated by mixing honey directlywith cane sugar.

The presence of many wheat starch

grains along with sugar cane fragments insome of the honey samples may indicate

that they are adulterated with minor qualitymolasse cane sugar syrup.

Microscopic analyses

The use of polarization microscopy (crossednicols 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 be

screened for parenchyma and sclereid cells,single rings and epidermis cells. Especially,the rings can be seen at the first glance.For proper identification of epidermis andespecially parenchyma and sclereid cells,it is better to use a higher magnification (400x) with the same microscopic technique orwith crossed nicols only. In this way thecharacteristic pits in the parenchyma 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 deviationand the range is shown in table III.

The number of parenchyma and sclereidcells in a typical white cane sugar samplefrom Nepal is 5 902 in 10 g. In an earlierstudy (Kerkvliet, 1982), it was found that in

various types of cane sugar originating fromdifferent countries, the number of singlerings ranged between 76 and 595 in 10 g and the number of epidermis cells between25 and 222 in 10 g. Parenchyma and scle-reid cells are present in a far greater num-ber, usually between 3 000 and 6 000 in 10 g. It was also found that cane sugars con-tained parenchyma, sclereid and epidermiscells and rings. From this observation it

seems highly unlikely that false-negativeresults are obtained in screening honeysamples for sugar cane fragments.

False positive results are highly unlikely.In the honey samples from beekeepers whoare under supervision of the NepaleseBETRESP project, no sugar cane fragmentswere found upon microscopic analysis.Chemical analyses also showed normal val-ues.

To make an estimation of the limit ofdetection of the presence of cane sugar in

honey, a minimum of about 30 parenchymaand sclereid cells in 10 g of honey can eas-ily be detected by screening the 1 cm2 area

of the microscopic slide of the sample. 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 percentcane sugar can be detected in a simple wayby this method.

CONCLUSIONS

Microscopic screening of honey samples forsugar cane fragments is an additionalmethod to detect even a minor addition to

honey of cane sugar, inverted cane sugarsyrup and ’honey’ from cane sugar fed tobees. As the investigated sugar cane (S offic-inarum) honey samples did not contain theplant cells from sugar cane described in this

study we may conclude that the presenceof sugar cane fragments shows that thehoney sample is adulterated. By countingthe sugar cane fragments an indication ofthe amount of cane sugar in the honey sam-ple can be obtained. The method is espe-cially usuful in the analysis of honey fromdeveloping countries and can be done withsimple equipment. In combination with theHMF value the microscopic method may dif-ferentiate between heating and adulteration.

Résumé &mdash; Diagnostic en microscopiede la falsification du miel par du sucre decanne 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 de régions (sub)tropicales. Le sucrede canne raffiné, le sucre de canne brut et lessirops de sucre inverti préparés avec dusucre de canne renferment toujours un grandnombre de cellules parenchymateuses destiges de canne à sucre, des cellules sclé-reuses, des anneaux isolés des vaisseauxannelés et des cellules de l’épiderme. Cesparticules sont absentes du sucre de bette-rave, du sucre d’érable et du sucre de palme.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 préparationmicroscopique de miel réalisée selon laméthode classique (Louveaux et al, 1978).Le résidu est repris dans 100 &mu;l d’eau. 10 &mu;lde ce volume sont étalés sur une lame, lereste sur une seconde lame et les 2 lamessont séchées. Les résidus sont montés dansde la gélatine glycérinée de Kaiser et étu-diés au microscope. L’étude a été faite enpartie au microscope polarisant avec uneplaque rouge I. Des cellules parenchyma-teuses, scléreuses, les anneaux et les cel-lules de l’épiderme ont été identifiés. Une

analyse quantitative est également possiblesur une préparation de 1 cm2. Les cellulesparenchymateuses, de forme généralementrectangulaire ou ronde, mesurent 50-100&mu;m de long et 40-60 &mu;m de large. Les cel-lules scléreuses, rectangulaires, mesurentenviron 100-200 &mu;m de long et 40-50 &mu;mde large. Les 2 types de cellules possèdentdes parois optiquement actives avec les cou-leurs de polarisation bleue (orientation nord-est/ sud-ouest) et jaune (orientation nord-ouest/sud-est). La surface des cellules estgarnie de nombreuses ponctuations (2-3 &mu;m de diamètre), chaque ponctuation pos-sédant une croix de polarisation ou unebande avec un entourage optiquement actif(jaune et bleu) (figs 1-3). Les anneaux desvaisseaux annelés ont 30-50 &mu;m de dia-

mètre, leurs parois une épaisseur de 4-5 &mu;m. Les anneaux présentent des couleursde polarisation bleue et jaune avec d’étroitesbandes rouges (figs 4-5). Les cellules del’épiderme, ±150 x 15 &mu;m, possèdent delongues parois ondulées et présentent descouleurs de polarisation bleu clair et jaune(figs 6-7). Dans les 10 miels falsifiés analy-sés, provenant des Philippines et du Népal,on a trouvé entre 455 et 7 845 cellules paren-chymateuses et scléreuses, de 8 à 1 025

anneaux et de 2 à 512 cellules de l’épidermedans 10 g de miel (tableau II). Cette méthodepermet de détecter des falsifications de mielslocaux avec seulement 1 à 2% de sucre decanne (ou de produits à base de sucre decanne).

miel / falsification / sucre de canne /

microscopie

Zusammenfassung &mdash; MikroskopischeMethode zum Nachweis von Verfäl-

schungen von Honig mit Rohrzucker undRohrzuckerprodukten. Es wird über einemikroskopische Methode zum Nachweisvon Verfälschungen des Honigs mit Rohr-zucker, Rohrzuckersirup oder Rohrzucker-fütterungshonig berichtet. Die Methode

wurde zur Untersuchung von lokalen Honi-gen aus (sub)tropischen Gebieten ent-wickelt. Weisser und brauner Rohrzuckersowie aus Rohrzucker bereitete

(Invert)Zuckersirupe enthalten immer einegrosse Anzahl Parenchymzellen desZuckerrohrstengels, Sklereidzellen, einzelneRinge von Ringgefässen und Epidermis-zellen. Rübenzucker, Ahornzucker undPalmzucker besitzen derartige Bestandteilenicht; auch in zwei Proben von Zuckerrohr-honigen (Saccharum officinarum) wurde kei-nes dieser Fragmente gefunden. Zum Nach-weis dieser charakteristischen pflanzlichenZellen wird ein mikroskopisches Präparatdes Honigs nach der klassischen Methodehergestellt (auflösen von 10 g Honig in 20 mlWasser, zentrifugieren, Rückstand mit Was-ser auswaschen, zentrifugieren). Der aus-gewaschener Rückstand wird in 100 &mu;l Was-ser aufgenommen, 10 &mu;l werden auf 1 cm2des Objektglasses aufgetragen und getrock-net; das übrige Volumen wird auf ein zwei-tes Objektglas aufgetragen und ebenfallsgetrocknet. Beide Rückstände werden zurmikroskopischer Untersuchung in Glycerin-gelatine nach Kaiser aufgenommen. ZurUntersuchung erwies sich die Polarisati-onsmikroskopie mit der Rot I Platte als vor-teilhaft. Identifiziert werden Parenchymzel-len+Sklereidzellen, Ringe und

Epidermiszellen; auch eine quantitative Aus-zählung auf dem 1 cm2 Präparat ist mög-lich. Parenchymzellen sind meistensrechwinklig oder rund, 50-100 &mu;m lang und40-60 &mu;m breit. Sklereidzellen sind recht-

winklig, ungefähr 100-200 &mu;m lang und40-50 &mu;m breit. Beide Zelltypen besitzenoptisch aktive Zellwände mit blauen (Lagenordost/südwest) und gelben (Lage nord-west/südost) Polarisationsfarben. Die Ober-fläche der Zellen hat viele Tüpfel (2-3 &mu;mDurchmesser) die ein Polarisationskreuzoder einen Balken mit optisch aktiver Umge-bung (gelb und blau) aufweisen (Abb 1-3).Einzelne Ringe von Ringgefässen habeneinen Durchmesser von 30-50 &mu;m, die Dickeder Ringwände beträgt 4-5 &mu;m. Die Ringe

zeigen blaue und gelbe Polarisationsfarbenmit schmalen roten Balken (Abb 4,5). Epi-dermiszellen haben Abmessungen von±150 x ±15 &mu;m und besitzen gewölbte langeZellwände. Sie zeigen helle blaue und gelbePolarisationsfarben (Abb 6,7). In 10 Probenvon (teilweise) verfälschten Honigen vonden Philippinen und aus Nepal wurden 455bis 7845 Parenchymzellen + Sklereidzel-len, 8 bis 1025 Ringe und 2 bis 512 Epi-dermiszellen pro 10 g Honig gefunden. Ver-fälschungen von lokale Honigen mit nur 1

oder 2 Prozenten Rohrzucker(produkten)sind mit dieser Methode nachweisbar.

Mikroskopie / Honig / Rohrzucker / Ver-fälschung

REFERENCES

Codex (1989) Codex Standards for Sugars (honey),Supplement 2 to Codex Alimentarius Volume III.Codex Alimentarius Commssion FAO/WHO, Rome,Italy

Czaja ATh (1971) Methoden der Lebensmittel-Mikroskopie und Lebensmittel-Ueberwachung.Umschau Verlag, Frankfurt am Main, Germany

Evenius J, Focke E (1967) Mikroskopische Untersuchungdes Honigs. In: Handbuch der LebensmittelchemieBand V, Kohlenhydratreiche Lebensmittel (J Schor-müller, ed) Springer Verlag, Berlin, Germany, 560-590

Jeuring HJ, Kuppers FJEM (1980) High performanceliquid chromatography of furfural and hydroxy-methyl-furfural in spirits and honey. J Assoc Off Anal Chem63, 1215-1218

Kerkvliet JD (1982) Het mikroskopisch onderzoek vanruwe en geaffineerde rietsuiker. De Ware(n)chemi-cus 12, 24-30

Louveaux J, Maurizio A, Vorwohl G (1978) Methods ofmelissopalynology. Bee World 59, 139-157

Sancho MT, Muniatequi S, Huidebro JF, Simal J (1991)Mieles des pais vasco.VII: Impurezas morfologicasmicroscopicas. Anal Bromatol 43, 173-183

Vorwohl G (1964) Die Messung der elektrische Leit-fähigkeit des Honigs und die Verwendung der Mess-werte zur Sortendiagnose und zum Nachweis vonVerfälschungen mit Zuckerfütterungshonig. Z Bienen-forsch 7, 37-47

White JW (1979) Spectrophotometric method forhydroxymethylfurfural in honey. J Assoc Off AnalChem 62, 509-514