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Measurementofhoneyquality

Introduction

Inthelast10yearshoneyproducers,especiallythoseinFrance,haveseen
increasedcompetitionfromimportedproductswhichdonotcomplywiththe
accepteddescriptionthathoneyisaproductharvestedbybeesfromplants
(andassuchitpossessestheplantscharacteristics);itis100%naturaland
nothingshouldbeextractedoraddedtoit.Insomecaseshoneyis
contaminatedbytheadditionofsugarandthesearchforcompetitivelypriced
productssometimesdrivescertainimporterstoacquirefalsifiedhoney(as
indicatedbythepresenceofstarchesandashes).RecentECregulations
(Directive2001/110)havesetnewqualityrequirementsforhoneyandarenow
beingimplementedinmemberstates(forexample,intheUKviaHoney
Regulations2003(SI2003/2243).

Animportantfactorinhoneygradingiscolourdesignation.Ascolourisan
importantcharacteristicusedbyproducers,packersandend-usersalike,its
measurementisvitalinqualitycontrolprocesses.Indeed,itisestimatedthat
75%ofindustrialusersincludecolourspecificationsintheirdesignations;
typicalusesareascolouringandbrowningagentsinvariousfoodproducts.

Otherparametersusedforspecifyinghoneyincludesugarcontent,moisture
content,waterinsolublecontent,electricalconductivity,freeacid,diastase
activityandhydroxymethylfurfal(HMF)content.

Measurementofqualityandpurity

Colour

Colourgradinghasbeenusedbythehoneyindustryformanyyears.In
naturalconditionthereisacontinuousrangeofcoloursrelatedtomineral
contentandfloralsource.Inadditionthereisaconnectionwithflavouraslight
colouredhoneyismildwhereasdarkertypeshavestrongerflavours.
Originallyasimpleopticaldevice,thePfundcolourgrader,waswidelyused,
whichcompareshoneywithafixedamberglasswedgeandthe
measurementswereincorporatedinvariousstandards.However,itis
recognizedbytheindustrythatspectrophotometryisjustasapplicableand
doesnotsufferfromtheinstrumenttoinstrumentvariabilityshownbyPfund
graders.Thusasimplemeasurementofabsorbanceat560nmenablesa
colourclassificationforhoneytobeestablished,asindicatedinthetable
below
ApplicationsNote:60
Technique:Spectrophotometry
ApplicationAreas:FoodandDrink







Spectrophotometry Page 2 of 5 Application Note: 60

Colournames(honey) Pfundscale
mm
Midrangeabsorbanceat560nm
Waterwhite <8 0.0945
Extrawhite 9-17 0.189
White 18-34 0.378
ExtraLightAmber 35-50 0.595
LightAmber 51-85 1.389
Amber 86-114 3.008
DarkAmber >114 >3.1

Diastasenumber

Honeyconsistsofmainlywater,glucose,fructose,sucrose,proteinsand
mineralsalts,plusseveralenzymes,includingdiastase.Thisenzyme
facilitatesconversionofstarchtomaltoseandisaddedbybeesduringhoney
production.Theactivityofdiastaseinhoneyisaffectedbystorageandis
sensitivetotemperatureincreaseandcanthusbeusedasanindicatorof
storagetime/freshnessandcontrolsduringprocessingofthehoney(HMF
contentisalsousedasanindicatorofhoneyquality).Althoughnaturallevels
arevariableinhoneysdependingonfloralsource,areductionindiastase
activityfromwhatisexpectedisausefulqualityindicator.

Legislationhassetaminimumlevelfordiastaseactivity;itshouldnotbeless
than8DNunits,where1DNunithydrolyses1mlof1%starchusing1gof
honeyfor1hourat37C.ThereferenceequationfromtheInternational
HoneyCommissiongivesadefinitionofdiastasenumberas:

Diastasenumber,DN=(28.2xabsorbancechangeat620nmafter10
minutes)+2.64

Methods

ThehoneyusedwasobtainedfromaknownindigenoussourceintheUK.
TheLibraS5isvisibleonlyinstrumentwithawavelengthrangefrom330830
nmthatmaybeusedforsimplemeasurementsat560nm.Italsohasa
heatedcellholderthatregulatesat37C,makingitidealforkineticsreactions
suchasthedeterminationofdiastaseactivity.

Tocarryoutthediastasenumbermeasurement,thehoneywasdilutedinpH
5.3acetatebuffertomakea5%solutionandthenmixedwith1%starch
solution.Themixturewasaddedtoacuvetteinthecontrolledtemperature
compartmentoftheLibraS5whichmaintainedthereactionofdiastasewith
thestarchsubstrateat37C.Sampleswerewithdrawnatperiodsof2.5mins
andaddedtoiodinesolution(1.3gofiodineand2gofpotassiumiodide,
dissolvedin100mlwater)whichwasthenmeasuredat620nm.

Spectrophotometry Page 3 of 5 Application Note: 60
Results

Colour

Theabsorbancereadingsforthehoneysolutionsusedwereasfollows:

Sample Abs560nm Classification

Honey 0.576 ExtraLightAmber

Notethattheuseofspectralmeasurementsforcolourismoreprecisethana
singlewavelengthasitcompletelyavoidsdependenceonagroupof
wavelengthswhichcouldbedistortedbyadulterationandomissionofrelevant
data.ScansarereadilyobtainedontheLibraS5.Approximationstopeak
areacalculationsafteroutputofacompletescantospreadsheetwouldenable
customisingthedatafurther.

Diastasenumber

Resultsofthestandardstarchsolutionhydrolysiswithnaturallyoccurring
diastaseinthehoneysampleareobtainedonthebasisthatasthediastase
reactionproceeds,thestarchconcentrationdecreasesandthisisindicatedby
theresponsefromtheiodinesolution.Theresultingabsorbanceoftheiodine
solutionat620nmwithtimeintothereactionisshownbelow(resultshave
beenplottedinExcelforclarity).

Honey diastase activity as measured by decrease in starch content of reaction mixture at

620nm. Linear best fit line and the accompanying regression equation is shown for comparison.
y = -0.0346x + 0.9178
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (mins)
A
b
s
o
r
b
a
n
c
e








Spectrophotometry Page 4 of 5 Application Note: 60
Forthishoneysamplethechangeinabsorbanceover10minutesfromthe
graphis0.37,correspondingtoaDN=13[calculatedfrom(0.37x28.2)+
2.64].Thisiswithinanormalrangeandwellabovetheminimumlegislative
requirements.

DiscussionandConclusions
TheseresultsdemonstratetheversatilityofthecompactinstrumentLibraS5
andshowhowitwillcontinuetheinvaluableroleofitspredecessorsforquality
controlinthefoodanddrinkindustries.Aconsiderableamountofdatamay
becollectedandarchivedfromarangeofsamples,requiringonlyfairlysimple
preparativemethods.Afurtheradvantageoftheunitisthesmallsizeand
convenientportabledesignwhichenablesittobeusedneartheprocess,for
exampletocheckcolourafterfiltration.NotethatotherLibrainstrumentscan
alsobeused,astheyallmeasureinthevisiblerange.

OrderingDetails

LibraS5withthermostattedcellholder 80-2115-01

Otherassaysforqualitycontrolofhoney

Measurementsofotherkeyparametershavebeenproposedbyregulatory
authorities.ThefollowingmayalsobeundertakenusingtheLibraS5and
otherinstrumentsfromtheLibrarange.

SugarContent
Individualanalysesofsugarscanyieldvaluableinformationaboutsourceand
floralorigins.AlargeamountofdatahasbeencompiledbytheSwissBee
ResearchCentretoshowtherangeofvaluesforseveralparametersfor
varioustypesofhoney.Forsugarsthisisparticularlydependentonfloral
originandthereforecouldbeusefulforcoordinationwithlabelling.Therefore
relevantmethodsare:
Glucose.Thisisanalysedusingglucoseoxidaseand4-aminophenazone,
andmeasuringabsorbanceat500nm.
Fructose.InthismethodfructoseisreactedwithtryptamineinHCl,at60C,
withcompletionin60minutes.Theassayisreadat518nmandshowsvery
lowinterferencefromothersugars.
Thecombinedfructoseandsugarcontentshouldbenotlessthan60g/100g
forblossomhoney.

Invertase
Likediastase,thisenzymeisalsosusceptibletoheatingandstoragefactors
socanbeusedalsoasaqualityindicator.Itisanalysedbyusingthe
substratep-nitrophenylD-glucopyranoside,withmeasurementsat400nm.

Spectrophotometry Page 5 of 5 Application Note: 60
Hydroxymethylfurfural(HMF)
Thiscanbeusedasanindicatorofhoneyqualityasitisasugarbreakdown
productandincreaseswithtemperatureandstoragetime.HMFhasan
absorbancemaximumat284nmandshouldbemeasuredovertherange250-
330nm;itsmeasurementthereforerequiresaUVrangeinstrumentsuchas
theLibraS12orS22.
Ingeneral,thereshouldbenotmorethan40mg/kgofHMFinhoney.