Understanding the Effect of Particle Size and Processing on Almond Lipid Bioaccessibility through

20 Pages·2017·14.74 MB·English

nutrients
Article
Understanding the Effect of Particle Size
and Processing on Almond Lipid Bioaccessibility
through Microstructural Analysis: From Mastication
to Faecal Collection
GiuseppinaMandalari1,2,MaryL.Parker2,MyriamM.-L.Grundy2 ID,TerriGrassby3 ID,
AntonellaSmeriglio1 ID,CarloBisignano4,RobertoRaciti1,DomenicoTrombetta1 ID,
DavidJ.Baer5andPeterJ.Wilde2,*
1 DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalScience,UniversityofMessina,
VialeSS.Annunziata,98168Messina,Italy;[e-mail](G.M.);[e-mail](A.S.);
[e-mail](R.R.);[e-mail](D.T.)
2 QuadramInstituteBioscience,NorwichNR47UA,UK;[e-mail](M.L.P.);
[e-mail](M.M.-L.G.)
3 SchoolofBiosciencesandMedicine,FacultyofHealthandMedicalSciences,UniversityofSurrey,
GuildfordGU27XH,UK;[e-mail]
4 DepartmentofBiomedical,Dental,MorphologicalandFunctionalImagesSciences,UniversityofMessina,
ViaC.Valeria,98125Messina,Italy;[e-mail]
5 U.S.DepartmentofAgriculture,AgriculturalResearchService,BeltsvilleHumanNutritionResearchCentre,
Building307B,Room213,BARC-East,Beltsville,MD20705,USA;[e-mail]
* Correspondence:[e-mail];Tel:[phone]
Received:19January2018;Accepted:9February2018;Published:14February2018
Abstract: Wehavepreviouslyreportedonthelowlipidbioaccessibilityfromalmondseedsduring
digestionintheuppergastrointestinaltract(GIT).Inthepresentstudy,wequantifiedthelipidreleased
duringartificialmasticationfromfouralmondmeals: naturalrawalmonds(NA),roastedalmonds
(RA),roasteddicedalmonds(DA)andalmondbutterfromroastedalmonds(AB).Lipidreleaseafter
mastication(8.9%fromNA,11.8%fromRA,12.4%fromDAand6.2%fromAB)wasusedtovalidate
our theoretical mathematical model of lipid bioaccessibility. The total lipid potentially available
fordigestioninABwas94.0%,whichincludedthefreelyavailablelipidresultingfromtheinitial
sampleprocessingandthefurthersmallamountoflipidreleasedfromtheintactalmondparticles
duringmastication. ParticlesizedistributionsmeasuredaftermasticationinNA,RAandDAshowed
mostoftheparticleshadasizeof1000µmandabove,whereasABbolusmainlycontainedsmall
particles(<850µm). MicrostructuralanalysisoffaecalsamplesfromvolunteersconsumingNA,RA,
DAandABconfirmedthatsomelipidinNA,RAandDAremainedencapsulatedwithintheplant
tissuethroughoutdigestion, whereasalmostcompletedigestionwasobservedintheABsample.
Weconcludethatthestructureandparticlesizeofthealmondmealsarethemainfactorsinregulating
lipidbioaccessibilityinthegut.
Keywords: almonds;particlesize;lipidbioaccessibility;microstructuralanalysis
1. Introduction
The behaviour of almonds in the gastrointestinal tract (GIT) may explain why almonds have
potentialhealthbenefitsandreduceriskfactorsassociatedwithtype2diabetes,cardiovasculardisease,
cancerandobesity[1–3]. Previousstudieshaveestablishedthatalmondcellwallsplayacrucialrole
inregulatingnutrientbioaccessibilityintheGIT[4,5]. Theterm‘bioaccessibility’isdefinedasthe
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Nutrients2018,10,213 2of20
proportionofanutrientorphytochemicalcompound‘released’fromacomplexfoodmatrixduring
digestionand,therefore,potentiallyavailableforabsorptionintheGIT.Usinganinvitroandaninvivo
study,wehaverecentlydemonstratedthattestmealscontainingalmondsofdifferentparticlesizes
behaveddifferently: thedegreeoflipidencapsulationaffectedtherateandextentofbioaccessibilityin
theupperGIT[6]. Wehavealsodemonstratedthatmasticationofnaturalrawalmondsreleasedonly
asmallproportion(7.9%)ofthetotallipidandwasonlyslightlyhigherforroastedalmonds(11.1%)[7].
Thelipidreleasefrommasticatedalmondswasincloseagreementwiththatpredictedbyatheoretical
modelforalmondlipidbioaccessibility[7,8]. Usinganinvitromodelofduodenaldigestion[9],itwas
observedthatadecreaseinalmondparticlesizeresultedinanincreasedrateandextentoflipolysis.
Novotnyetal.[10]conductedafeedingstudyinhealthyadultstodeterminetheenergyvalue
ofalmondsasarepresentativefoodfromagroupforwhichtheAtwaterfactorsmayoverestimate
the energy value. They showed that only 76% of the energy contained within almonds (based on
theAtwaterfactors)wasactuallymetabolised[10]. Furthermore,whencalculatingthemetabolisable
energy(ME)ofwholenaturalalmonds,wholeroastedalmonds,choppedalmondsandalmondbutter,
it was demonstrated that the number of calories absorbed was dependent on the form in which
almondswereconsumed.
Basedonthesefindings,theaimsofthepresentworkwere: (a)toinvestigatethemechanisms
responsibleforthelossinobservedinvivometabolisableenergycomparedtothatcalculatedfrom
nutrientcompositionusingtheAtwatergeneralfactors;thiswasperformedbymicroscopyinpost-GIT
faecal samples; (b) to carry out microstructural investigations on freshly artificially “masticated”
almondsamples,todeterminetheparticlesizedistributionofthe“masticated”samplesandtheextent
oflipidreleaseafteroraldigestion; and(c)tofurthervalidatethemathematicalmodelpreviously
developedtopredictlipidreleasefrommasticatedalmonds[8].
2. MaterialsandMethods
2.1. AlmondandFaecalSamples
Fouralmondtypesallwithbrowntestapresent(naturalrawalmonds, NA,roastedalmonds,
RA, roasted diced almonds, DA and almond butter, AB) were provided by the Almond Board of
California. SmoothunsaltedABwasindustriallyproducedbygrindingunskinnedroastedalmonds.
Itcontained(asperlabel): fat(50%,ofwhich4.7%weresaturated),totalcarbohydrates(25%,ofwhich
12.5%weredietaryfibreand6.2%weresugars),protein(15.6%). Faecalsampleswerecollectedfrom
humanswhowereparticipantsinastudytomeasurethemetabolisableenergyofthealmonds[11].
Thisfeedingstudywasacrossover,randomizedcontroltrial. Volunteers(10menand8women)were
fedthe5distinctfeedingregimes(control,NA,RA,DAandAB)aspartofahighlycontrolleddiet.
Duringthefeedingperiods,allmeals(usinga7-daymenucycle)forthevolunteerswerepreparedat
theBeltsvilleHumanNutritionResearchCentre(theCentre)andMondaythroughFridaybreakfast
anddinnerwereconsumedattheCentreundersupervisionoftheresearchinvestigators. Lunchand
weekendmealswerepreparedattheCentreandpackagedforconsumptionoff-site.Foodsforallmeals
andsnackswereidentical(exceptfortheformofnut). Foodforallmealswaspreparedbyweight,
tothenearest1g,toproducedailymenusprovidingarangeinenergyfrom1600kcalto4000kcal.
Volunteerswerefedtheenergyneededtomaintaintheirbodyweight(bodyweightwasmeasured
eachmorning,MondaythroughFriday)andadjustmentstotheamountoffoodconsumedwasmade
byincreasingordecreasingtheamountofallfood,proportionately,suchthatthecompositionofthe
dietwasidenticalforallvolunteers,independentoftheenergytheyrequiredtomaintainbodyweight.
A total of 42 g/day of each form of nut (NA, RA, DA and AB) was consumed daily with half the
amountconsumedatbreakfastandtheotherhalfconsumedatdinner. Forthecontroldiet,theamount
ofallfoodswasincreasedproportionatelysuchthattheenergycontentofthecontroldietwasdesigned
tobeequaltothe4fourdietsthatcontainedthecontrolfoodsplusthenuts. Volunteerswererecruited
from the area around the Centre and were screened to insure they met the study criteria. Briefly,
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subjectswerehealthyindividuals(nottakinganymedicationsorsupplementsthatmightinterfere
withstudyoutcomes)withoutdentalordigestiveconditions. Atthebeginningofthestudy,themean
(±SEM(Standarderrorofthemean))ageofthevolunteerswas56.7±2.4year,theirmeanheightwas
170.2±2.1cmandmeanweight88.6±5.6kg.
Fromeachofthe18volunteers,faecalsampleswerecollectedfromthebeginningandendofeach
of5distinctfeedingregimes(control,NA,RA,DAandAB)(eachtreatmentperiodlasting3weeks).
Followinga14-dayadaptationtoeachofthe5feedingregimes,thestudysubjectsreceivedbluedye
capsulestomarkthebeginningofaoneweekexcretacollectionperiodandasecondbluedyecapsule
tomarktheendofthecollectionperiod.
ThestudyprotocolandinformedconsentformwerereviewedandapprovedbytheMedStar
Health Research Institute and the associated faecal samples were registered with the QIB Human
ResearchGovernanceCommitteeinDecember2016.
2.2. SimulatedOralDigestion
The aim of this procedure was to simulate the chewing of the almond meals in the mouth.
Chewing is the initial step in the digestion process and this procedure was designed to simulate
boththesalivaryamylaseactivityandthemechanicalbreakdownofthefood. Fouralmondsamples,
NA,RA,DAandAB(25g),wereminced3timesusingamincer(Lexenmincer,Windermere,UK)
tosimulatethemechanicaloralbreakdownofthemeal. Thereafter,12.5mLofSimulatedSalivary
Fluid (SSF) at pH 6.9 (0.15 M sodium chloride, 3 mM urea) and 900 U Human Salivary Amylase
(HSA)dissolvedin1mLSSFwereaddedtothemincedalmondsorthealmondbutter[12]andmixed.
Thisprocessproducedapasteofequalratioofsolidtowaterascalculatedfromhumanchewing[13].
2.3. ParticleSizeDistribution(PSD)
The particle size of the samples before (AB) and after simulated oral digestion (NA, RA, DA
andAB)wasmeasuredusingmechanicalsieving. Briefly,22.5gofeachsamplemixedwithSSFwas
loadedonastackofsieveswith9aperturesizes: 3350,2000,1000,850,500,250,125,63and32µm
(Endecotttestsieveshaker,EndecottsLtd.,London,UK).Thesampleswerewashedwithdeionized
water, shaken for 15 min and washed again, thus ensuring separation of the particles. The sieves
werethendriedinaforced-airovenat56◦Cfor6h. Thebaseswerelefttodryat100◦Covernight
(about15h),whichpermittedcompleteevaporationofthewater. Thesieveswereweighedbefore
loading the sample and then again after having been oven dried. The dried fractions retained on
eachsieveandthebasewereexpressedasapercentageoftheweightofalmondsbeforesimulated
oralprocessing.
2.4. LipidReleaseafterOralProcessingandMathematicalModel
Lipidextractionfortotalfatdeterminationonallsamples,beforeandafteroralprocessing,was
performedwithaSoxhletautomaticSoxtec2050extraction(FOSSAnalytical,Hilleroed,Denmark)
usingn-hexaneasasolvent[5]. ForAB,itwasassumedthatthecontinuousoilphasewasbioaccessible
andthereforetheaimwastomeasuretheadditionallipidreleasedfromthealmondparticlespresent
inAB.ThealmondparticleswereseparatedfromthecontinuousoilphaseofABbycentrifugation
(REMI Elektrotechnik LTD., Vasai (East), India (13,000× g, 15 min) before and after chewing and
thelipidcontentoftheparticleswasdeterminedasdescribedbelow. Thefatpresentinthepellets
(almondparticles)wasalsodetermined: thepelletwaswashed5timeswithwarm(37◦C)distilled
watertoremoveanyfreefatreleasedfromthecells,thenseparatedbycentrifugationandquantified
byn-hexaneextraction. Thelipidpresentwithintheremainingwetpellet(theoreticallyinsidethe
almondcells)wasextractedbytheBligh&Dyer[14]method. Briefly,almondparticleswereextracted
inchloroform/methanol/waterintheproportions1:2:0.8andthelipidwasthenquantifiedinthe
chloroformlayer.
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2.5. ResultsofLipidContentWereExpressedasaPercentageofDryWeight
Lipid release was predicted from the measured particle size distribution by sieving and the
previouslymeasuredaveragecelldiameter,36µmusingthemethodofGrassbyetal.[8].Thethreshold
diameter below which 100% release would be achieved was 54 µm, particles below the threshold
werenotincludedinthecalculationsoflipidrelease. Thespreadsheetprovidedassupplementary
informationwasmodifiedtoacceptparticlesizedatafromsievingaloneandtoaccountfortheparticles
abovethethresholddiameterthatwererecoveredonthe63µmsieve(50µm<p<100µm).
2.6. MicrostructuralAnalysis
Portionsofeachalmondsample(NA,RA,DAandAB),beforeandaftersimulatedoraldigestion,
wereincubatedinthechelatingagentCDTA(1,2-Cyclohexylenedinitrilotetraaceticacid)(50mMCDTA,
pH=7)at4◦Cforaminimumof4weeks[15]. Thistreatmentweakensthepectinlayerinthemiddle
lamellabetweencellssothatindividualcellscanbeseparatedfromlumpsoftissuebygentlepressure.
Theseindividualcellswerethenobserved,unstained,bymicroscopy(bright-fieldorpolarizingoptics),
orafterstainingwithSudanIV(0.1%SudanIVin1:1acetoneand70%ethanol)tovisualizetheiroil
content. TheCDTAtreatmenthaspreviouslybeenfoundtopreventmicrobialgrowthandtoretainthe
oilfractionintheformitexistsinthecellsofthekernel,eitherasindividualoilbodiesinNAoras
largecoalescedoilglobulesinRAwhicharecharacteristicoftheroastingprocess[15]. Freshsections
ofrawandroastedsampleswerenotusedbecausethesectioningprocesswasfoundtoreleasetheoil
fromthedamagedcellssothatthespatialinformationwaslost.
Initialobservationsweremadeonfaecalsamplesfrom3randomly-chosenvolunteersonmaterial
storedinCDTA[15]. Itwasfoundthatthismethodwasnotoptimalassomeofthefreeoilcontentrose
tothesurfaceandlargerlumpsoftissuesankintheCDTAmakingitdifficulttoobtainarepresentative
sample. However,itwasausefulpreliminarysteptohelpidentifytherangeofplantstructuresthat
survivedpassagethroughthebowel. Theseincludedwheatbranlayers(primarilyaleurone,thecells
of which are similar in size to almond tissue) and brush hairs, vascular tissue, xylem and tannin
bodyinclusions.
Subsequentinvestigationsonsamplesfromvolunteerswithmeasuredhighfaecalfatcontent
weremadebymixingasmallsampleofthefrozenfaecalmatterdirectlyonamicroscopeslidewith
theoilstainSudanIVtominimiselossofcomponents.
Formicroscopy,sampleswereexaminedandphotographedusinganOlympusBX60(Olympus,
Southend-on-Sea,UK)microscopeandProgRes®CapturePro2.1software(Jenoptik,Jena,Germany).
2.7. StatisticalAnalysis
Results of lipid release from mastication were expressed as mean ± standard deviation
(SD) of four independent experiments and analysed by one-way analysis of variance (ANOVA).
The significance was assayed by using the Student-Newman-Keuls test using the SigmaPlot 12.0
software(SystatSoftwareInc.,SanJose,CA,USA).Statisticalsignificancewasconsideredatp<0.001.
3. Results
3.1. ParticleSizeAnalysis
Theweightofmasticatedalmondretainedonthesieves,presentedasapercentageoftheoriginal
weightofthemasticatedalmond,wasplottedagainsttheaperturesizeofeachsieve. Theaverage
PSDsforthedifferentalmondsamplesisshowninFigure1. NA,RAandDAhadverysimilarPSDs
withmostoftheparticleshavingasizeof1000µmandabove. Ontheotherhand,ABboluscontained
mainlysmallparticles(<850µm). ThePSDsofNAandRAweresimilartotheonesmeasuredfor
boluses from our human study [7], demonstrating that our simulated oral processing was a good
alternativetohumanmastication.
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Figure1.Particlesizedistributionsbymechanicalsievingofalmondboluses(n=2).Theweight%of
allmaterialrecoveredinthesievebase(<32µm)isgivenatsize=0.01µm.
3.2. LipidReleaseafterSimulatedMasticationandPredictedLipidRelease
Thereleaseoftotallipidasapercentageoftheoriginallipidcontentofeachsample(53.4%,w/wfor
NA,54.1%,w/wforRA,55.6%,w/wforDAand50.1%,w/wforAB)aftersimulatedmasticationis
reportedinTable1. Inagreementwithpreviousdata[15],between8.9%and11.8%oftheoriginal
lipidintheNAandRAsamples,respectively,wasreleasedasaresultofmastication. Thehigherlipid
releaseinroastedDAcomparedwiththatdetectedinRAcouldbeexplainedbytheincreasedsurface
area in DA,whichwereroastedafterdicing. ForAB,tocalculatethetotalavailablelipid, wehad
to determine the additional lipid released from almond particles in AB as a result of mastication.
ThelipidreleasedfromparticlesinABfollowingchewing(6.2%)wascalculatedas%lipidcontentof
theremainingintactalmondtissueafterthefreelipidinthecontinuous-oilphase(48.2%oftotallipid)
andtheavailablelipidassociatedwiththealmondparticles(39.6%oftotallipid)hadbeenremoved
(seeSection2.4). Therefore,thetotallipidavailablefordigestioninAB,obtainedbycombiningthe
availablelipidduetoinitialprocessing(continuousphaselipidplusavailablelipidassociatedwith
theparticles)andtheadditionallipidreleasedfromtheremainingintactparticlesduringmastication,
was94.0%.
The values for predicted lipid release are presented in Table 1: a close agreement with the
measuredlipidreleasewasobtainedwithNA,RAandAB.Themeasuredlipidreleasewashigher
thanthepredictedreleaseinDA:thisisprobablytheeffectofroasting.
Table1.Lipidreleasedduringmastication(%)andpredictedlipidrelease(%)fromnaturalrawalmonds
(NA,n=4),roastedalmonds(RA,n=4),dicedalmonds(DA,n=4)andalmondbutter(AB,n=4)dueto
sampleprocessingandmastication.Valuesrepresenttheaverage±SD(standarddeviation).
Almond LipidReleasedDueto PredictedLipid TotalLipidPotentially
Meal Mastication(%) Release(%)* AvailableforDigestion(%)**
NA 8.9±0.7 9.6 8.9±0.7b
RA 11.8±1.1a 12.6 11.8±1.1b
DA 12.4±0.8a 9.6 12.4±0.8b
AB 6.2±0.4 6.4 94.0±4.6
*Sieving,averageofn=2;**Referredto%oftotallipid;ap<0.001vs.AB;bp<0.001vs.AB.
Nutrients2018,10,213 6of20
3.3. MicroscopyExaminationonAlmondBaselineSamples
Observationsonbaselinesamplesareessentialtocharacterizethedifferencesinmicrostructure
betweennaturalrawalmondtissue,almondtissueprocessedbyroastingandalmondtissueroasted
andgroundtobutter. Thesedifferencesarerelevanttothebehaviourofthealmondmaterialduring
chewingandsubsequentlytothefateofthealmondtissueinthedigestivetract.
3.3.1. NA
Individual whole cells separated from raw almond tissue by CDTA are shown unstained in
Figure2aandstainedwithSudanIVtolocatelipid(Figure2b). Thecellsaresmall,lessthan50µm
in diameter and tightly packed, with well-defined cell walls. The appearance and distribution of
the protein bodies and lipid in unstained cells after CDTA treatment is consistent with previous
observations[15]thatthelipidisstillwithinoleosomes,surroundingtheproteinbodiesasinmature
rawalmondsnottreatedwithCDTA.InSudanIV-stainedmaterial,thesurvivaloftheoleosomesis
demonstratedbyanevendistributionoflipidwithineachcell(Figure2b).
Figure2.CDTA-separatedcellsofbaselinenaturalrawalmonds(a)unstained(b)lipidstainedwith
SudanIVandroastedalmonds(c)unstained(d)lipidstainedwithSudanIVshowinglipidcoalescence
inthecellsfollowingroasting.
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3.3.2. RA
Theeffectofroasting,inbothwholeanddicedalmonds,istoliberatethelipidfromtheoilbodies,
whichthenformslargelipiddroplets,asseeninbothunstained(Figure2c)andstainedcells(Figure2d).
Itispossiblethatindividualcellsinchoppedalmondsmayachieveahigherinternaltemperaturethan
thoseinwholeroastalmondsduetothegreatersurfaceareaexposedtoroasting.
3.3.3. AB
ThebaselineABsampledifferedconsiderablyfromtherawandroastedbaselinesamplesinthat
mostofthecellsofthekernelsareruptured,releasingthelipid(andothercellcontents)toformapaste.
Cellfragments(walls,proteinbodies,nuclei,testaandsomesmallintactclumpsofcells)aresuspended
inacontinuouslipidphase(Figure3a),whichwasstainedwithSudanIV(Figure3b).Fragmentsofthe
browntestaarerichincalciumoxalatecrystals(Figure3c).Proteinbodies,liberatedfromthecellsbythe
grindingprocess,retainthedimpledsurfaceimpressionscreatedbythesurroundingoleosomes(Figure3d).
Theothermajorparticulatecomponentsofthealmondbutterarefragmentsofcellwalls(Figure3d).
Figure3.Almondbutterbaselinesampleshowingcellularfragmentssuspendedinalipidphase(a)
asmallgroupofkernelcellsfromwhichthelipidhasbeenreleased(b)thelipidphasestainedwith
SudanIV(c)fragmentsofbrowntestacontaininglargecalciumoxalatecrystals(d)proteinbodiesand
cellwallfragmentsinthelipidphase.
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Additionalinformationonthecomponentsofthealmondbutterstartingmaterial,particularlythe
proteinbodies,wasobtainedbyfirstde-oilingthebutterwithchloroform/methanol(1:1). Thematerial
couldthenbeviewedmoreclearly(Figure4)unstained,orstainedwithdiluteaqueoustoluidineblue.
Although the butter looks finely ground, it does contain some multicellular particles in the range
150µm–1mmasillustratedinFigure4a. Followingde-oiling,theproteinbodiestendedtoclump
togetherbutsmallcrystalsofcalciumoxalatearevisiblewithinthelargerproteinbodies(Figure4b–d,
blackarrows)underpolarizingoptics. Alsovisiblewithinthelargerproteinbodies,afterstainingwith
dilutetoluidineblue,aresphericalgloboidbodiesintheformofsmallspheres(Figure4d,bluearrow).
ThesegloboidshavebeenshownbyEDX(EnergyDispersiveX-ray)analysistoberichinphytin[16],
themainsiteformineralstorageinseeds. Itshouldbenotedthattheproteinbodiesandtheircontents,
releasedfromthecellsbygrinding,arefarmoreaccessibletodigestiveenzymesthatthosestillwithin
cellwalls[17].
Figure4. Almondbutterbaselinesamplede-oiledinchloroform/methanol:(a)Thebuttercontains
clumpsofkernelcellsandtestafragments;(b,c)individualproteinbodiesformclumpswithsome
containingsmallcrystalsofcalciumoxalate(blackarrows)visibleunderpolarisingoptics;(d)protein
bodiesstainedwithdilutetoluidineblueshowinganoxalatecrystalandphytingloboids(bluearrow)
foundinthelargerproteinbodies.
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3.4. MicroscopyExaminationafterSimulatedOralDigestion
3.4.1. ChewedNA
Thesizerangeofmulticellularparticlesoftheunstainedchewedrawsampleatlowmagnification
is shown in Figure 5a. Lipid which has been expressed by the chewing process is present as
droplets of different sizes. In this sample, which has undergone artificial chewing and storage in
CDTA, the lipid droplets appear to be a single lipid phase. However, preliminary observations
on fresh volunteer-chewed NA showed that many of the larger lipid drops were in the form
of water-in-oil-in-water (WOW) compound emulsions but the internal water droplets tended to
coalesce over time. It is possible that storage in CDTA was less than optimal for stabilising the
artificially-chewedsampleandthatanycompoundemulsionsthatmayhaveformedduringchewing
werelostduringstorage.
SeparationofthecellsinthelargerparticleswasfacilitatedbyCDTAandshowedthatmanyof
thesecellsareundamaged(Figure5)andthatthelipidisstillpresentinoleosomes.Thiswasconfirmed
afterstainingwithSudanIV(Figure5c,d)tolocatethelipidinundamagedcells,damagedcellsand
freelipiddroplets. Comparedwithotherparenchymatoustissue,thecellsofalmondseedsarevery
smallandsomanycellsescapetheshearingandcrushingforcesduringchewing,bothsimulatedand
inthemouth.
Figure5. ChewedwholerawalmondsstoredinCDTA:(a)multicellularparticlesofalmondtissue
surroundedbyreleasedlipiddrops;(b)individualundamagedcellsseparatedfromthelargerparticles
contain lipid still within oleosomes; (c) Sudan IV staining showing the even distribution of lipid
inoleosomeswithinwholecells; (d)lipidreleasedfromdamagedcellscoalescesintolargerdrops
withoutinclusions.
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3.4.2. ChewedRA
Roastingisknowntomakethealmondtissuebrittlebut,asintherawsample,therearemany
particles consisting of intact cells in an emulsion of released lipid (Figure 6a). Lipid drops adhere
tothesurfaceoftheseparticles(Figure6b). Althoughtheparticleslargelycontainundamagedcells,
thelipidineachcellhascoalescedasnotedfortheunchewedroastedsamples(Figure6c). Athigher
magnification(Figure6d)manyofthereleasedlipiddropsareseentoconsistofawater-in-oil-in-water
(WOW)compoundemulsion. Thefactthatthesecompoundemulsiondropspersistedduringstorage
inCDTAsuggeststhattheyaremorestablethanthoseinchewedrawalmonds. Apossibleexplanation
isthatduringtheroastingprocesswhenthelipidbodiescoalesced,additionalinterface-stabilising
compounds,suchasproteins,werereleasedfromthecellsandthesethenbecomeactiveatthesurface
of the internal water phase drops, effectively preventing them from coalescing. The mechanisms
underlyingthiseffectcouldbethesubjectoffuturestudies.
Figure6.ChewedwholeroastedalmondsstoredinCDTA:(a)multicellularparticlesofalmondtissue
surroundedbyreleasedlipiddrops; (b)lipiddropsstainedwithSudanIVadheretotheparticles;
(c)lipidisreleasedfromoleosomesduringroastingandcoalesceswithintheundamagedcells;(d)many
ofthereleasedlipiddropsareintheformofawater-in-oil-in-wateremulsionwhichpersistduring
storageinCDTA.