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A Fluorescent
LectinTestfor
Mold in RawTomatofuice
S.J.POTTS,
D.C.SI-AUGHTER,
ANDI.F.THOMPSON
ABSTRACT:Fungal (mold) contamination is an important indicator of low quality raw product in the processing
tomato industry. A quantitative lectin assaywas developed that was less expensive, faster, and more precise than the
industry standard Howard mold count. This assay,based on a fluorescent-labeled lectin isolated from wheat germ,
had a selective affinity for the chitin in fungal cell walls. Assayvalues were correlated with mold contamination for
4 fungal species: Alternaria alternata (r'z: 0.91), Cladosporium herbarum (r'z: 0.75), Fusarium orysporum (rz :
0.97), and Stemphylium botryosum (1'z: 0.99). Combining all 4 species, the lectin assayhad a strong coffelation (r2
: 0.76) with a linearized Howard mold count.
KeyWords: filamentous fungi, biosensor, tomato, lectin, mold detection
lntroduction
fluorescentlabels,and their nomenclaturederivesfrom the
name of their source.TherearenumerouscommerciallyavailTHE
I in raw productat inspectionstationsand in processedprod- able lectinsthat bind polymersof N-acetyl-D-glucosamine.
uct in qualitycontrol laboratories.
At the inspectionstations,23 Stoddardand Herbertson(1978)utilized fluoresceinlabeled
kg of fruit from each24metricton truckloadof processingtoma- lectinsto detecthuman pathogenicfungi.Patel(1992)usedflutoes arevisuallyinspectedfor defects.Tomatoeswith visible oresceinisothiocyanate(FITC)labeledlectinsto observemold
signsof mold are weighedto obtain a percentageof decayed in processedfoods.He testedseveralchitin-bindinglectins,and
fruit on a massbasis (PTABI996).At the processor'squality found that a lectin from wheatgerm agglutinin(WGA)had the
control laboratories,mold is quantifiedby the Howardmold strongestbinding to fungal cell walls and the leastamount of
count (HMC)method (AOACl9B4),wherea smalldrop of ho- nonspecificbinding to tomato cells.He observedconsiderable
mogenizedjuice is inspectedusinga microscope.In the HMC autofluorescent
at simisignal,tomatocelltissuethat fluoresces
method, 2 slidesof 25 fields each areviewed,and the percent- lar wavelengthsas the fluorescentprobe. Patel and others
ageof fieldscontainingmold are recorded.An accurateHMC (1993)usedbiotinylatedlectinsand streptavidinlabeledmagtakesup to 30 min to conduct.Overthe last severaldecades, netic particlesto separateand concentratemold sporesand
many attemptshavebeenmade to replacemanual gradingand yeastsin fruit juices.
the Howardmold count with a lesssubjectiveand lesslabor inThe goal of this researchwas to developa rapid method of
tensivemeasurement,but no method hasbeen accurate,rapid, quantifyingmold in raw tomatojuice usinglectins.Ideallythe
and simpleenoughto use at inspectionstationsor in quality measurementwould havea linearrelationshipto mold content
control laboratories(Iarvisand Williamsl9B7;Gouramaand at levelscommonlyfound in raw tomato juice, havea lowervariBullerman1995;Cousin1996).Despitethe difficultiesand limi- ability than the HMC, and givea similar responsefor different
tations of the HMC (for example,Williams lg68; Iarvis and oth- fungal species.The test should alsobe simpleto conduct and
ers l9B3),it remainsthe universalstandardfor mold assess- useinexpensivereagents,and giveresultsin the samelengthof
ment almost 90 yearsafter it was first introduced (Howard time asthe HMC.
l9l l).
Resultsand Discussion
Chitin is an important structural component in fungal cell
walls,but absentfrom plant tissue.The detectionof mold based
on the chemicalisolationand quantificationof N-acetyl-D- Tomato tissue autofluorescence
glucosamine,
a breakdownproductofchitin, hasbeenproposed
The fluorescenceemissionspectramaximumfrom freshtoas an alternativemold measurement(Rideand DrysdaleI972; mato juice occurredat a longerwavelengththan the maximum
Iarvis 1977;Lin and Cousinl9B5).Jarvis(1977)found a coeffi- for FITC labeledlectin when excitedat 490nm (Fig. l). Microcient of variation(CV)of around20%for this method.Although scopicobservationsshowedthat strongesttomato autofluoresthe high performanceliquid chromatographybasedisolation cencewas observedin the fibrovascularbundles,the stem
method is too slow and labor intensiveto be utilized as an indus- cells, and the skin cells.The wavelengthdifferencebetween
trial replacementfor the HMC, a more rapidmethodthat detects the emissionmaximum and the excitationmaximum is known
chitin could havecommercialpromise (Cousin1996).
as the Stokes'shift, and representsthe loss of energydue to
Lectinsarenaturally occurring proteins or glycoproteinsthat moleculardissipation.The Stokes'shift for tomato tissuewas
bind to specificcarbohydrates.
They arebecomingincreasingly consistentlyaround B0nm for excitationin the visible range.
valuableasmolecularprobes,includingthe labelingof cell-sur- Fortunately,FITC,like most commonlyusedfluorescentlabels,
face componentsin tissuetyping (Lis and Sharon1986).Hun- has a considerablysmallerStokes'shift of only 15 to 20 nm.
dredsof lectinsfrom microbial,plant, and animal cellshavebeen Longpassfilters are normallyused in fluorometeremission
identified,but most commerciallyavailablelectinsare isolated measurements,so the photomultiplier tube would detectthe
from plant seeds.They are availablewith various enzymaticand autofluorescentlight. However,we used a bandpassfilter,
CALIFORNIA TOMATO INDUSTRY MONITORS MOLD LEVELS
346
:ounNAL oF FooD scr€NCE-Vor. 65.No.2. 2000
@ 2000 lnstituteof Food Technolooists
where only a narrow band of light, centeredat 520 nm, would
be detectedby the fluorometer,to eliminatethe tomato autofluorescentsignalshownin Fig. I and alsoobservedby Patel(1992).
Table | -Fungal species variability in average HMG levels for tomato
iuice with an average spoiled tissue mass of O.75yo
Howard Mold Count(o/o)
Species
Howard mold count results
The Howard Mold Count scoresfor the juice samplesin this
study rangedfrom 0% to 100%for all mold speciesexceptC.
herbarumwhich had a maximum HMC of 96%.The average
(acrossall dilution levels)amount of mold for each species
wasO.75Vo
spoiledtissueby mass.The average(acrossall dilution levels)HMC scoreslbr eachspecieshowever,rangedfrom
alow of 37.4Tofor C. herbarum to a high of 64.2Vo
for A. alternafa (Table1).
The HMC resultswere nonlinearwith spoiledtissuedilution
level (Fig. 2) due to field saturation where additional fungal
myceliain an alreadypositive field did not increasethe readings.Considerablevariability,particularly at the intermediate
spoiledtissuelevels,wasobservedbetweenthe HMC scoresobtained by the different quality control laboratories(QCL).The
variabilitywaslower at both the zeroand maximum spoiledtissuelevels,sinceno valuecan be under 0% or over 100%respectively.The overallaveragecoefficientof variation (CV)between
the averageHMC scoresof all four quality control laboratories
was35%.The HMC scoresobtainedby the quality controllaboratorieswerewell correlated(Table2), with the best agreement
occurringbetweenlabs3 and 4 (r = 0.97).
Lectin assay results
In contrastto the HMC assay,the lectin assayresultswerelinearwith spoiledtissuedilution levelresultingin the following coefficientsof determination:A. alternata12: 0.9I, C.herbarum12
= 0.75,F.oxysporumrz : 0.97,S.botryosum12= 0.99,all species
combined12= 0.59,Fig.3. This linearity is important, both because,unlike HMC,it allowsthe lectin scoresto directlyindicate
actualmold levelsmakingthem easierto compareand because
it makescalibrationsimpler.The nonlinearHMC methodwasat
saturationat 27ospoiledtissuefor 3 of the 4 species.A KruskalWallisone way analysisof variancetest showedthat the precision of the lectin assaywas significantlybetter (cr= 0.02)than
the precisionof the HMC assayperformedby any of the quality
controllaboratories(Table3).
Althoughthe spoiledvolume dilution will give accuraterel-
Alternaria
alternata
Stemphylium
botryosum
Fusarium
oxysporum
Cladosporium
herbarum
64.2a
53.1ab
46.2bc
37.4c
18.3
t o ./
14.9
14.5
(o= 0.05).
withthesamegrouping
" Meanvalues
letterarenotsignilicantly
ditferent
Table 2-Gorrelations
between average Howard mold count values
obtained by 4 quality control laboratories
1.00
noa
0.91
0.91
QCLl
OCL2
QCL3
OCL4
1.00
0.85
0.85
Table 3-Average
coefficient
WGA lectin mold as$ay
1.00
0.97
of variation
Evaluator
(GVf values for HlllO and
Ave. CV*
QCLl
50.3%a
45.6o/oa
42.OY"a
21.60/ob
7.2o/oC
WZ
QCL3
QCL4
WGA LectinAssay
'CV valueswithlhe samegroupingletter not
(u = 0.02)difierent
are
significantly
ative results (that is, 0.5%level has exactlyone quarter the
m o l d a s 2 T o l e v e l )i,t i s u n l i k e l yt h a t t h e r e i s t h e s a m ed r y
weight of mold massin the original 2Vomold levelsfor different fungal species.For example,the averageHMC at 0.5%
spoiledtissuedilution was 94 for A. alternata and40 for C. herbarum.Sincethe HMC givesan indicationof fungalbiomass,
we used these scoresto adjust for varying amounts of fungal
biomassin the undiluted contaminatedjuice. Becausethe
HMC is by nature nonlinear with high variability,we developed a linearizedHMC scoreto comparewith the lectin assay.
The HMC scoresof the 2 quality control laboratories(3 and 4)
100
100
s
i
-80
E9
-t=r tc
o
o60
:o.80
E"
s.z
Standard Deviation
60
EE ao
EP
ov
320
lr
c
5
E
o
=40
tt
(E
420
0
o
0.5
1
1.5
2
Mold Level (% Spoiled Tissue)
Fag. | -Tomato tbsue autofluor€scence and FITG labeled WGA lectin
emission spectra for a 49O nm excitation.
Fig. 2-Howard mold count results for tomato juice infected with
Stemphylium botryrosum.
Vol. 65, No. 2, 2000-JOURNAL OF FOOD 5C|ENCE
347
Lectin Testfor Motd
o
g
tr
D
o
500
o
o
G
400
o
(,
300
E
o
(,
o
o
o
=
l!
0.5
0
1
1.5
2
Mold Level (% Spoiled Tissue)
Fig, 3-Lectin
aasay y$ mold dilution leyel.
o
o
(,
th
o
=
E
o
N
o
o
c
1
1.5
2
o
0.s
(%
Spoiled Tissue)
Mold Level
Fig. 4-Linearized
Howard mold count for tomatoiuace anlected with
Stemphylium botryoaum.
o
6
I
o
o
=
tt
o
l!
6
o
tr
100
200 300 400 500 600
Fluorescence (Relative Units)
Fig. S-Lectin
700
assay uersus linearized mold count.
348 rounNAL oF FooD ScrENCE-Vor.
6s,No.2.2ooo
which had the bestprecisionamongblind replicatemeasurementsand the highestcorrelationbetweenlaboratorieswere
averagedand used as the "true" Howard mold count for mold
levelsin the study.Four mold levelsfor C. Herbarum (that is,
0% through l% spoiled tissue)and 3 mold levelsfor the remaining fungalspecies(that is, 0% through 0.5%spoiledtissue)were regressedagainstthe spoiledvolume to developline a r i z e dH M C m o d e l sf o r e a c h s p e c i e sT
. h e s em o d e l sw e r e
then usedto predict linearizedHMC scoresabovethe linear
rangefor eachspecies(Fig.4). The linearizedHMC scoreswere
then regressedagainstthe lectin assayreadingsresultingin
the following coefficientsof determination:A. alternata12:
0.91,C. herbarum12: 0.76,F.oxysporum12: 0.97,S.botryosum 12: 0.99,all speciescombined12: O.76(Fig.5).Thesere_
sults show that the lectin assaywill give generallycomparable
resultsto HMC in the linear rangefor eachfungal organism.
Differencesin responseamongspeciesare either intrinsic in
the lectin assay(thatis, speciesdiffer in amountsof chitin, or
in number of exposedchitin bindingsites,Sharmaand others
1997;Cousin 1995),may reflectthat speciesdiffer in ratio of
fungalmassto spoiledtissuemass,or HMC scoresmay differ
f o r s i m i l a r q u a n t i t i e so f d i f f e r e n tm o l d s p e c i e s( T a b l el ) .
E i s e n b u r g( 1 9 5 2 )o b s e r v e dh i g h l e v e l so f v a r i a b i l i t ya m o n g
fungal specieswhen comparing the relationship between
H M C a n d s p o i l e dv o l u m e i n i n d i v i d u a lt o m a t o e s .B a t t i l a n i
and others (1996)also observedfungal speciesvariabilityin
b o t h t h e r e l a t i o n s h i pb e t w e e nH M C a n d s p o i l e dv o l u m e a s
well as the relationshipbetweenHMC and ergosterolcontent.
The lectin assayrequired 120min for a group of 12 samples,
or an averageof I0 min per sample.This is one-thirdthe time required for an accurateHMC analysis.The total cost of reagents
usedin the lectin assaywere$0.60.The costof the lectin assayin
a commercialapplicationcould be reducedfurther if the reagentswerepurchasedin largequantities.
Microscope observations
WGAlectin bound stronglyto the myceliaof all 4 species.
Strongestbinding occurredat the hyphal tips and in the septa
(cross-walls),
althoughtherewasgenerallyan adequatecoating
of older wall surfaces.However,it did not bind the asexualconidia of any of the species,with the exceptionof E oxysporum,which
exhibitedsomelectin binding to macroconidia.
Conclusion
TESTFORMOLDWASDEvELOPED
A RAPIDAND INEXPENSI\ts
.{lLwhich has the potential to replacethe Howard mold count
for testingof mold in raw tomato juice.The testis linear,with 3 to
7 times better precisionthan the Howard mold count for the four
fungal speciestested.Lectinsare inexpensive,and the assay
takesaround l0 min per samplewhen analyzedin groupsof 12,
one-third the time required for an effectiveofficial Howard mold
count.\Mth binding time reductions,it has the potentialto be a
much fasterassayand can be automated.Correlationswith mold
count resultssuggestthereis an acceptablylow variationacross
the 4 fungal speciestested (Alternaria alternata, Cladosporium
herbarum,Fusariumorysporum,and,Stemphyliumbotryosum).
Futurework will involve a largernumber of fungal speciesand
will look more closelyat the quantitativedifferencesbetween
species.
Work to reducethe bindingtime from 40 min and to automatethe procedurefor usein inspectionstationsand quality
control laboratoriesis alsoneeded.
Materials and Methods
Fungal cultures
(varietyHeinzBB92)
processing
Ripe,defect-free
tomatoes
ed from 220nm to 800nm (5 nm bandwidth).An emissionspectrum wasalsorecordedfor a FITClabeledWGAlectin solution excited at 490nm, nearits reportedexcitationmaximum.
werewashedand surfacedisinfectedfor 10minutesvnth a2%o Howard mold count procedure
Fourtomatoprocessors
eachreceived60 tomatojuice samsodiumhypochloritesolution.Eachof four setsof 20 fruit was
placedonto a sterilewire meshin a sterilecontainer.Cultures ples,consistingof threeblind replicatesof the five spoiledtisof Alternaria alternata,Cladosporiumherbarum,Fusarium ox- sue dilution levelsfor eachof the four fungal species.These
ysporum, and Stemphyliumbotryosuim,isolated from tomato processorsperformeda Howardmold count,viewingtwo slides
fields,weregrownon potatodextroseagarat22 'C for up to 21 for eachsample,and the percentof positivefieldswasrecorded.
days.Eachfruit was prickedjust below the surfacewith a sterile knife and inoculatedwith one of four fungal pathogens. Lectin assay
Anothersetof60 (5 dilution levelsx 4 speciesx 3 replicares)
The fruit were placedinto an incubator at 26 "C for two to five
days (dependingupon fungal growth rare)with 16 h of light juice sampleswasusedin the lectin assay.One of the 60 tubes
into a 50 ml cenand B h of darknessper day.The fruit remainedin the incuba- wasrandomlyselected,l0 ml of juice.pipetted
tor until the fungi spoiledapproximatelyz%o(by mass)of the trifuge tube, alongwith 40 ml of Tiis buffet pH 8.3 (Brooksand
others 1997),centrifugedin a swingingbucket centrifuge (IEC
tomatotissue(Fig.6).
The spoiledvolume (Battilaniand others1996)was cut ClinicalModel)at 5000RPM(2260I for I min, and 40 ml of the
from eachfruit in a setand addedto unspoiledtissuefrom ad- supernatantwereremoved.Highlyreactivenonspecificbinding
ditional ripe, defect-freeprocessingtomatoes(varietyHeinz siteswereblockedwith 200p1of 30%bovine serum albumin
BB92)and comminuted for 40 s in a blender (Waringmodel (SigmaChemical,St.Louis,Missouri),and 50(lof lmg/ml FITC
CB-6,Hartford,CN) to obtain3.6 kg of juice containing2% labeledWGAlectin (EYLabs,SanMateo,California)was added.
spoiledtissue(by mass).A separateset of B0defect-freepro- The tube was shakenfor 40 min in the dark on a wrist action
cessingtomatoeswerealsocomminutedfor 40 s in the blender shakerto allowadequatebinding to occur.Lectinbuffer (40ml)
to obtain 3.6 kg of juice containingno spoiledtissue.The to- was added,and the tubes centrifugedat 5000RPM (2260g) for
matojuice with 2%spoiledtissueand thejuice with no spoiled I min. The supernatantwasremoved,leavingonly the cellspeltissuewerefiltered(640micron pore size)and combinedpro- leted at the bottom. The centrifugingand washingstepwas reportionallyto obtainfivejuice sampleswith spoiledtissuedi- peatedonce.The washedcellswere resuspendedin l0 ml of
lution levelsof 0.0%,0.25To,0.5Vo,
1.0%,and 2.0%(by mass). buffer beforefl uorometermeasurement.
A fluorometer(TurnerModel450,Barnstead/Thermolyne,
Eachdilution levelwas subdividedinto 40 ml replicatesubsamples,placedinto sealabletubes,autoclavedat l2l "C for Dubuque,IA) was used to quantify the presenceof FITC labeled lectin,equippedwith a 490nm excitationbandpassfilter
20 min, and then storedat B'C for up to threeweeks.
(TurnerNB490,l0 nm bandwidth) and a 520 nm emission
bandpassfilter (TurnerNB520,l0 nm bandwidth).The 520nm
Tomato tissue autofluorescence
Cleanmold-freejuice wasdiluted and placedinto a scanning emissionbandpassfilter wasusedto block out the autofluoresfluorometer(HitachiF-2000,SanJose,CA).The excitationwave- cent signalat wavelengthslongerthan the fluoresceinemission.
lengthwassetat 490nm and the emissionspectrumwasrecord- The fluorometerwascalibrateddailywith a standardsolutionof
FlTC-labeledWGAlectin. The readingswere checkedwith this
solution everythirty minutes during the study,to correctfor
small levelsof instrumentdrift. Juicefrom the preparedsample
waspouredinto a 5 ml borosilicateglasscircularcuvette(Fisher
Scientific,Pittsburgh,PA),and a readingrecordedon the fluorometer.The cuvettewasemptiedand rinsed,and the cuvette
relilled with sample.A total of five readingswere averagedto
obtain the fluorescentscorefor eachsample.
The precisionof the lectinassayand of the HMC assaywere
evaluatedby calculatingthe CVacrossblind replicatemeasurements conductedon eachmold dilution level/speciestreatment. The usual analysisof varianceassumptionsof a normal
distributionand equalvarianceacrossgroupswerenot valid for
the coefficient.of variancevaluesanalyzed.Both the modified
Levene(Levene1960;Brown and Forsythe1974)and Bartlett
(Bartlettand Kendallf946)testsshowedthat the varianceofcoefficient of variation valueswere significantly(c1: 0.01)different betvveenassays.The Kruskal-Wallis one-way analysisof
variancetestwasusedin placeofthe usualanalysisofvariance
techniquebecauseit doesnot rely on theseassumptions.
Fig. 6-lnfected tomato truit showing growth of Fusarium
orysporum. The cone-shaped spoiled tisgue was cut frcm each
truit and weighed.
Microscopic observation
Samplesfrom the assaywere observedunder an epifluorescentmicroscope(Zeiss,standardmodel),equippedwith a
fluoresceinfilter set (Excitationmodel BP490,Emissionmodel
LP 520)to checkboth lectin binding to mold cellsand nonspecific bindingto tomato tissue.
Vol.65,No.2, 2000-JOURNALOF FOOD9CI€NC€ 349
Lectin Test for Motd
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We ile grateful to Eunice Tan and Robert Neilson, in the Biological and Ag. Engineering
Department at UC Davis, for their assistmce in conducting this study, to David Glahn md
the Depiltmenr of Cell Biology md HmanAnatomy at UC Davis, for the use of the scanning fluorometer, and to R. Michael Davis in the Planl Pathology Depiltment at UC Davis
for the donation of the cultures. We would also like to thanl the California Processing
Tomato Advisory Board for ihe funding they provided for this study
Authorsare with theBiologicaland Agricultural EngineeringDepartment,
UniuersityofCalifornin,Dauis,Dauis,CA95616.Directinquiries to author
Slaughter(E-mail: dcslaughter@ucdauis.
edu).