Coast Malaysia Condiments on Dentine Pulp Ti$sues Preparations

Tekspenuh

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ANNALS OFlv.fICROSCOPY VOL4.May 2004

Morphological Changes Re af,ed to Adherence Properties

of

Se~ectedEast

Coast Malaysia Condiments on Dentine Pulp Ti$sues Preparations

FaridBin Che Ghazali, WanAbdul MananWan Muda and Jamarnddin MatAsan

School of Health Sciences) Universiti Sains Malaysia) Health Campus. Kubang Kerian) KelantanJ Malaysia16150.

ABSTRACT

Local condiments oonsumption is akeyconstituentofdiets throughout the eastcoast ofPeninsu1ar Malaysia. Little evidenoe-basedknowledge islmown ofthe morphologicaIchanges thatmayimpingeon oral health due to consumption ofthese localcondiments.VPSE~,a :qewhighlydeveloped model of scanning electron microscope with simplifiedpreparation ofthe tested material before use(Billy, 2000), has never beenutilized before to study the effects ofthese locally available Mala:ysian condiments.This studyisanattempt to pioneerfuelltilityofhigh-resolutionmicrOscoPY especiallywith VPSEMinelilcidating aneedtoqualitate these condlments by scientific investigationthus; hoping to improvethiscommercially viable pr'Oducts presentquality.Atpresent their quality attributes arebas~dmainlyon flavor and smell.

KEYWORDS:Anchovies and:fishsauces;,soyand tomato condiment;. lrigh-resolutionrnicroscopy~d Variable pressure scanning electronmicroscopy (vpSE:M).

INTRODUCTION .

Fermentedfoods are essential components ofthe dietina number ofdeveloping countries, and are consumedeitherasmaindishesor as condiments(Steinkraus,1996).Fermented condiments are·product offelIDentationevolvedfor thedevelopmentoftaste or aroma; it oftenres~tedin enhancecl-nutrition, stabilization ofthe originalraw materials, and detoxificationofanti-nutrientfactors. Several fennented condimen-m relied on the introduction ofvarious organisms such as from;tb.e Bacillus species):includin~

Bacillus natto andB.subtilis. Withthe selected ingredientand additives, the finished products are ofavery local character and usuallywillexhibit,sensory properties reiulting from uniqueselectedflora andritual processingtecbnologie,s applied inSlnallscale, home-basedfermentations (BeaumontM, 2002). Dooto the lackofscientific and

tee

ologicalknow~how;theseco]Dinercially viable locallyproduced fermented condiments are generally evaluatedonthebasis ofsubjective qualitativeattributyssuch as odour and flavour.

Kelantanis a stateinthe east coastofPeninsularMalaysia. Amongits traditional foods that remain to be part ofthe regular diet ofa large segment ofthe population, is atype ofcondimentknownloc?1J.y as 'budu'. Thelavious enjoymentoflocalcondiments consumptionremainsakeyconstituentofdietstbroughout manyparts ofthe east coastMalaysia. Most oftbese condiments were preparedbyincubatingthe fish or shrimpinhighconcentrations ofsalt andunderhighhumidityat ambient temperature overseveralmonths.

Generally; the fish sauces were only made from selectedfish species such as anchovy anj. sardine (Jung..

Nim Park,et all 2001).Fishand other types ofmanne-derived food are good sources of long-chain polyunsaturatedfatty acIds. atty acids are essential elements for neural developme- -tin the infantin utero anddUl1ngthefustfew years after

birth

(Uauy,Mena,,&Rojas, 2000). Theparticulai" fatty acid incolporated in. e brain and retina ofthedeveloping infantisdocosahexaenoic acid or DHA. Since the human body lacks the enzymes to manufacUn"e fatty acids, DHAmustbe suppliedbythe diet, orproducedin vivofrom diet-delived fatty acidprecursors such aslinolenic acid

38

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ANNALS OFMICROSCOPY Vol 4, May 2004

Other condiments consumed are the traditionally fennented soybean condiments, such as soybean paste and soy sauce,thatar~commonly consumedbyotherpeopleinSouth East Asian countries (Mongkolwai, Assavanig, Amnajsongsiri, Flegel,&Bhumiratana, 1997).Soysaucehasbeenknown to contain strong anti oxidative and free radical scaveIiging activity (Yamaguchi, N. and Fujmaki, M. 1974).

Although.thenitrogencompoundswere emphasizedasone ofthe importantfactors onantioxidative activity

·ofsoysauce, the maincomponents ofbrown colorproductsinsoy sauce werereco~as melanoidin, the endproductsofMaillard reaction process, which's also strongly suggested as a compolUld holding-

antloxidative activity (Gapsoon Moona,et aI.,2002) .

Itis well known that mineralised human tissues such asitsbones andteethreacttovarious physical and chemical stimuli. These reactions are often observed quite differently tothatofother tissues and organs ofthe body. The denrineralisation ofbone matrix and offullycalcifiedbone have been studiedby

Boo~oyd(1964),. and Thorogoodand Ora)) (1975), whereas the mineralisationresearchwas C<?nducted ontoothtis~uebyWatson (1960) andDecker(1973). However studies.haveyet

to

be carried outto observe the relationship beu.veenthe consumption oflocal.sauceswiththenaturalprocess ofdemineralisation and loss ofctystallites from1hemineralisedtissues1hatmightbeinducedandattnbutedbyregularconsumption ofthese local delights. Thus little is known ofthehisto mOlphological changes that might occurs andthat mightgive rise to highincidences oforalhealthis-~such as white spotsand~carie.,toothformations thatmay be associated with. the consumption ofthe condiments. -

Design andsetting:

~ This cmrent studywas designedtoinvestigatethe predisposingrole ofselectedlocal condim,ents onwhitespots fonnation onhealthy mineralised teeth. structure.

• The study was conductedinthe Health campus ofUniversiti SainsMalaysia tbatis situatedinthe ,east coast ofPeninsular Malaysia. Theprimaryobjective was to assess the ultra..motphology -chapges that may occur and correlate the .findingwiththe adherence properties ofthe selected

condiments.

• To register a national morphological database oneffectsoflocal consumption ofthesesauces especiallyto tb;e mineralisedtissues ofthe humanbody.

• "Itishope thatinsightsgainedfromthisstudyWill:furtb.erupdate~owledge ofregular consmnptioDS ofthe condiments. The data obtainedwill also be useiUl innutritionaI evaluationofsegments ofthe

population consumingthese food products .

Eqlerimental metbodology

Fish anchovies, tomato sauceketchup andsoyasaucewere randomly selected from the.wide range 6fcommerciallyavaiklbleproductlistforthepilotstrid~Only:freshvarieties oftheidentifiedlocalcondiments masedon expiry dates will beused. Each portion oftheidentifiedcondimentswas placed into various labelled 1QOmlbottle containers. These specimen containerswillthen be.fixedtoa variable lowtor~e

speedvialr:otatoratroomtemperature; (this willhelp to improve thepenetrationofthe condimentsingredients Intothemineralisedtissue). Phandthe expirydates ofeachcondimentwillberegularlynoted Themmeralised tmsue will be initially disinfectedwitb.~<?lgargle, then directly immersed into each labelledcondimerrt containers. Regular'daily changes offreshportions

of

thevarious condiments were subjectedtothe . ;-ij'ralisedtissues. Each day the various morphological changes were examinedmacroscopicallyusing a

SLereomicroscope;by when every eachInorphological change observedwas clerked andreQ9rded. Under n~-)rrnalcircumstancea complete demineralisation ofatoothstmcture will hypotheticallybe achieved within.

th~

Iftre

fourthtothefifthw~ek:,hencethe IDOlphologicalchangeswillbestudied bylatefifthweekOnthe 1ifi11~eek,each mineralised tissues was cheinicallyfixedfor afull duration ofoile daywith:fullstrength K9moVsky's solution(pH 7.0) following whichthey wererinsedgently inRingers solutions. (Ifneeded, the

~h~mical1yfixed specimens were then x-rayedto checkforradiolucency.an4radio..opaclty). The specimen's

Wereprepared as ground section where~eywere sectioned as unstained sections usingadiamond saw

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ANNALSOFMICROSCOPY VOL4~May 2004

microtome at selected 201mspecimenthickness increments. Each ground sections were thengrin~and polished for optical nucroscopeobs~rvations. The ground sections were observed under the LEICA stereomicroscopeandvariable pressure scanning ,electronmicroscope,. The ground sectionwas initially washed withphosphatebuffers solution No osmificationwas caniedout. The preparationwas examined under the LEO-VPSEM using its peltier cooling sub-stage at 35 to 48 Pascal pressure at an accelerating voltage of

is

kV andaround9lll:tll to 15mmwor~g distance. The tooth ground sections they was examined initially from thell' occlusal surface, thentothe pulpal border andclerked Photomicrographs images ofthe sections were taken and then transferredtoLeicaimaging workstation to enable accurate analysis ofthe deminemlisedareas.Theresultant imagewas measuredand colour-codedin accordanceto

various histogram classes. .

The umnersion experiments"were based onvarious time~rvals record~dat 24-Hours, 72 hours to 4days and finally 4 weeks and after. Visual stereo and VPSEM charting ofvarious morphological chaiIgeswillbe recorded -

RESULTS

1. pH and immersionincondimen.ts experiments

" The results from thisstUdyindicate that the pH ofthe condiments used were consistentthrough the period ofimmersion experiments. The meanpH are as follows' Tomato sauce meanpH=4.3. Anchovies saucemeanpH=5.97. Soya beansauce mean pH=5.I.

24...Hours, immersion

(i) Stereomlcroscopeobservatio~. .

-Nochanges or defonnation was observed on the sutface occlusal morphology ofthe entire too$

crown immel'&ed m the various:condiments selected - (ilJ Varlablep eSSUf'eSEMobs,ervatioDs.

Thethreadlike structure observed spiraling at someregions ofthe mid dentine inthe fish anchovies

condiments immersionexperimentpreparatioDS. .

. 72 hours to 4daysim ersion

(h) Variable pressur.e SEM bservations.

Infish anchovies.condiments immersion experiments, the finding obsetved was consistent withthe 24hours finding observation. The threadlike structure suggestive ofputative odontoblastprocess was still observedinmid-dentine region.

Inthe tomato sauce condimeD:ts immersion experiments) at the coronal end ofthe pulp chamber the pulpaltissue seemstobe totally dissolved and absent ofanyjSoft tissues remnants. The mid dentine tubules waswell defined.and also clean ofany putative, structures. At 2000X magnifications, the dentinal tubules was observedas shallow:furrows with bluntborders and edges, no shalp deri:larcationedges was observed.

Such an observation gives this region ofllie dentine an appearance, dentinal tubules ofa wider diameter width. Attheenamel dentine junction (EDJ), literatures have reported that the dentinal tubules here are filled with.peritubularde~tinewithitsmainmineral.component ofca1ciumphospbate.. Although oonsidered amorphous, this peritubUIardentin~was reported to be crystalline octacalsium phosphate (Be 'kovitz;

Holland andM:oxbam; 1992).Inthis experiments when observation under theVPSEM at the enamel dentine junction was conducted, the sample insertedintomato sauce condiment shows no crystalline perltubolar dentine present. What was observedat the EDJ was the folding waves/oontour ofthe gaps or

40

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:4NNAl;S:OFMlCROSCOPY

the intertubular dentine between the tubules.Itis hypothetically suggestive thatthisf~lditlg,way~' ~@

indicationthat demineralizationprocessisoccurringattbatsite.

~/d-: bin~oJf.sauGecbndime t&.Hnm.~exp~~~]lulp:tiS:sue Wf1s~SerYeat01bepreSentifJnd

~llmta'¢t-as wNholeoonne.~ve.tisSl!e.:bui!.d11t1yqlii 's-a'Iit dlsplaGP.•,d,fbM·if8:a0tdaJ:~~lhetenbl@o.aaen: Jt~;

~theoor0nwrpaitofth~ pulVilhamhendt~sug~~e;])Ulp8i 'tiSsue~ ~]~isj'PldeEgOinga§Jo'MP(oceSS­

ibf.disorgaW2ati6'nand!displac~etfu~ .~r&ldibg~wav..es·weietabse~:d1lJ/tMmwrll1buhtt··demtine{·~ ry ad0e.was·WS·tpreseiltitJr.t1'letOOU10sJrhe.go~saht.e· :theQ"taJ)Hoon4imentthatiW~Jflts$.Vedl'te~enta.t MUsJifne$J1te1tv.alirutberolall$a1tub;yJ.es:alth<IDgliaiprep~hs&rQm$e~ersi()n·~xp~t&was

;gent1y{waslied:befer~p1acemeilt ontG~'smdof:thdp-eltier:sfagl{camht!f<ttfNPSEMAbsemati.coW3:J

! -

FignreI .

=Stetean:nQ{Qst.opepho~iiliQr~gmp ·offb:othenam.posWpn:neisedmthe.:seWG~4~p.dimentsr

i~':.,:~:Ster.eomiclroscQpeiob~av.DuOl1S'~f~.I·:~

I~;': !: -.Sur.face:bcclusalfhorpholog)f;Qf~e:tOvllUJ'l.i,fUWlliwas'Ooserve<i1lilsmg.tbe'steroonrlorpscope at.16*.

! 'DmajOisauce;specini:ens:sh(jwedsigris~ofdemin.~i:aJisati<mtas ..e.ly_as';week4:i1!tiinmersiQn~ :TheyJwete softintextureandflabbylikem!llatUre, this,phenomenon;was·noyPbservedinthe-Oft!ooeondimelitimmetsibn samples.

-(i"~ ':'Yariable:preSsur~' S~~:ooseH'atio'ns..,·;

,',l'h¢useofthel8awmiCIotomeleads,to:the{ormatien:ofsmearJayerimpre~teslonfthe:clitsurface

~.oftheteoth.The sni'eaflayet:s'were~bickandyrsible,seems especially;mth~tomato sauce;specimens.

lOb.setvatioll ofits:stnfacetopographywas.a1mdstimpossible"althoughattemptrwas- - ~aPetoremdve the :smearlayerwiththe:useDf3S.phosphoricacid'fo~ .:minute... The.specimens~werethen."gently.fracturedto

m~bidthecut·smface.. Thefraotnririgtechniquewasioonducted:gentlywith~pr€cision hammering withthe fuse()famaUet:.ObS"~rvation was madeusmgtlie.vPSEM~t9ttimworkingdis~ooat 4'JPascalptcssure with the (backscatteri.Qgelectron) BSED mode. TheresUlts

can

bes~.s~(tas·belc,.w; ..~ .: ,.' .. , -.

a. therewas no smeat layer observed around the fractured surfaceinallspecimens.Smearlayer . wasonly--ob~ervedon~~cutstnf-ace;andnfthe1:breegroups.ofspecimens'"'Condimehts samples;

the leasteoated-was~e'surfaceofspeQimens~imme1sed-with Soyabean ketchup:-:

.... .~... ;

:The·~m.at9-~mwespe.ci:t;neDSwere covil'eclWiithawelh·4efinedboatingibelievedtobetheadherenee e'fthe~atroe

and.:Sinear

llayer-ooth¢lsmfacearea:ofthe speoimeas. This]Nasmtewsting·to-be bbser:v.edand noted as the phenomenon ofcoating~as-lio1 obs~ed.t(}occUl!'dn ethersP~cimens iWi&o~er.conditilents althoughallofthemhave been thorougblywashed underrunning tap water andthenakedeyedo not show anypresent ofa coating or acquired pellicle like surface ofwhat ever sort ofCQlourto bepresent.·

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ANNALS OFMICROSCOPY DISCUSSIONS

VOL4, MaY' 20f)4

The mean average Ph

of

our selectedfish.anchovies condiment budu, is 5..97. Attbi-s average Ph,:it seemsthat ofthetbreeselectedcondiments usedinthisstudy,the fishanchovies showed the least and slow characteristic cha:nges observed macro and micro-morphologically. From past studies the mean average Phvalue offish sauces producedinthe South East Asia region was cited within 5.4to5.8 'Ph level, Myanmar fish-sauce isatan average meanPh value of6.23, Laotian4.90

and

Chinese fish saucesat6.15 (ReD.,eta11 199~,Fujii,et al., 1992). The pHvalue offish sauce was also notedto ·be ofahigher reading then soyasaucewheretheyranged from4to5pH. Itwillbe interestingtostudyifthevariousmo1phological changesobserved·

ere

will

be

similarlyobservedwhen immersedintheabovecondiments. Thefishsauce was also reportedtobeof highsaltconcentration(15to 25%w/w), hencenotrecommendedto be consumedinhighquantities (Aryanta,etal.t 1991). '

The tooth preparations used in this experiment, are'frotnfreshly'surglcallyremoved wisdomteeth gained from consented patients at the government dental clinic Jalan Mahmood Kelantan. Thetooth preparation comprises ofenamel",deritine

an4

coronal pulp tissues.Thistooth model was selected.as the mineralisedpreparationfor not only ofthe anatomical features but duetothe factthatthisstmctures ofthe tooth are.' directinstancecontact especIallylinked to adherence principals WIththeselected condiment once consumed. The variousstereoand VPSEMtnorphologicalchanges'reported shouldbe database beneficialtotheoralcare worker.Thisinfonnationcaninytease awarenessandscientifie-basedoralheath promotiontowardsbetterlifes1yleea~ghabits especiallyfromthe consumptions of ocalipride condiments from locally available home basedindustries.

Theear~ydemineralization stage,observedunderthestereomicroscopewas identifiedas welldefined whitish chalky areas or islandpatchthatareusuaGyvis"hIe on the occlusal margin and ridges ofthe tooth crown preparations.Thiswhitish chalky island is a signofan eaxlycariesfonnationidentified as white spots and this is related to demineralization ofthetoothhardest tissue, the enamel. We believed that this pvocess ofenameldemineralization is

maxIDPzed

bythe fourthweekespecially.in tomato condiments immersion experiments. However,ifthe soft tissue such as the pUlpal tissueisallowedtobe exposed and thus allowing the oondimentsto adheretoit,then asearlyaswithin24hoursthere will be totaldisintegration ofthe softtissue leadingtoanon-vital stage ofa former vital tissue structure.

The variable pressure SEMteclmique (ormicrostru:clura1 observationrequires n.o chemical fixation process,

and

thusreduces the preparation time andpossible artifacts, as compared withthe age ofthe conventional scanningelec1ronmicrosoop~ The localcondim~tsselectedhere is aseries ofwidelyavailable commercial home basedproductsthat were pl:eparedbyritual and anecdotal. So far, withno scientific based microscopy eff()J;ts haveeverbeenrecorded to enhance andhencetoregulate further its quality. To the knowledge oftheprese~tresearchertheclosestqualitycontrol evaluation is limited to subjective . qualitative attributes suchasflavor and odour. Hence, high-resolutionmicroscopymonitoringandevaluation investigationsespeciallywith the usage ofVPSEMiShighlyreconnnendedand as shownhere haveproven necessary for improvingqualityand for advocacy.

Small-scalehome industriesbasedfood fermentation technologiesinMalaysiaand its neighboring countriesisbyritual haveevolvedthrough years ofexperience (or village-art methodologIes), ratherthan tbroughnovelscientificbreakthroughs. Many ofthese small-scale manufacturers are therefore; illiterate thus reluctantto accqjt changes andmodifytheir fennentation prC!cesses. Upgrading the quality and safety offennentedfoods, while reducing theirproduction cost andmaintaining their authenticity andumqueness, isofutmostimportanceandhigh...resolutionmicroscopy·candoitspart?ere.

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ANNALS OFMICROSCOPY CONCLUSION·

Vo14JMay2004·

Consumptionoflocal condiments is a delight that iscommerciallyviableanddemograp;h.ic specific that colors one"s culture. Although pH and mineral oontentisbelievedtohave playedhypothetically a significantroleinthe changesobserv~'it was mostlikelythattheadherence, viscosity andpermeability capabilities ofeachs~lectedcondimentstothedentinepulpcomplexpr~parationsthat havelead

to

the

mmphological changesobserved "

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Boothroyd, B. (1964); The problem ofdt:mineralization"inthinsectionsoffullycalcifiedbone.J.Cell

BioI., 20: 165..173 .

Decker,ID'. (1973), Fixationeffectson the fine :structure ofenamel crystal-matrix relationships.J.

Ultrastrlfc.. Res.,44: 58-74.

Fujii, T. Nikkuni; S.andIida, H. (1992). Chemical compositionand putresciblepotential ofcommercial 4GShottsum", Jap'anese fish sauce. Nippon Shoku.hin KogyoGakkaishi. 39, PP 2013;. 706.

'Gapsoon, Moona,- MhliaLe~b".. YoungsunLee and Gassinee Trakoontivakom (2002). Main component.

ofsoy~aucerepresenting antioxidative. activity. International CongressSeri~s.Vol~e 1245, November, Pages 509-510.

Jung-Nim.Park, Yuki Fukumoto, Eriko Fujita, Tadayoshi Tanaka, TakuyaWasbio, Soichil'oOtsuka,Tetsuji Shimizu, KatsukoWatanabe andHirokiAbe.. (2001). ChemicalCompositionofFishSaucesProduced inSoutheast and EastAsian Countries..Journal ofFood Composition andAnalysis. Volume 14, Issue 2, April, Pages 1. 3-125.

Leach, SA.&.(1983)Demineralis~tion ~dRemineraUzation ofthe Teeth: OxfordUniversityPress.

Mongkolwai, T.)Assavanig,A. Amilajsongsiri, C., Flegel, T.Wand Bhuminltana, A. (1997).T~hriology

transferfors.ma1land medium soy sauce fenrientationfactoriesin.Thailand: acoDSortnnnapproach FoodResearqhlnternational, 30 (1997),pp2013,563.

ReD, H. Hayashi, T4 Endo, H. :andWatanabe, E. (1993)4 Characteristics ofChinese and Korean soy arid fish sauces on the basis oftheir free amjno acid composition.,Bull. Jpn. Soc. Sci.Fish..59,PP

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Silwood, C.1.Prinz, J. Grootveld, M and. Lynch, E. (2000). Protective EffectsofH~fIurotitinate against demineralisation ofthe hard dental tissues. Proceeding oflADR meetingWasbington4

Steinkraus,K..H.(1996).fu:HandbookofIndigenousFennentedFoods,MarcelDekker,NewYork,pp

. 2013;274. .

Thorogood., P.

v., J.e.

Gray, (1975), D'emineralisationofbone matrix: Observations from electron . microscope and electronprobeanalysis.Calcif. TissueRes" 19: 17-26;

Thylstrup,A. Leach, S. A. Qvist,V.(1987). Dentine andDentineReactionsintheOralCavity. Oxford University Press."' .

Uauy, R,Mena, P: ,and Rojas,C~,(2000).Essential fatty, acids ine~lylife: s1ructural and functional role.

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492.

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ANNAhS'CJFMICROSCOPY

'Yamaguchi, N. andFujimaki~M: (1974).Stu~eson browning reactionproductsfro~ed1tlcingi1l:igittS

and ammo acids: Part 14.Antioxidativ;e activities ofpurifiedmelanoidinandtheir comparison with .those.,oflega1:;antioxidants-'vournalojFoodSci?nceatfcPleehrl:crrlogJJ(Japan}2f;<p!6~:

The autho:r:s would liketoacknowledge that the fimding ofthis researchproject was supportedbya USMshort~tenn.grant:3045IPPSKl613 1217.And thankDrEndang Purwani forpro~cJiJfg fr~

surgically removed tooth that was usedinthis experiment

Below~theVPSEM1?h~ti)nrlcrQgr-apb$iljIust.t;ationsof~edentinesladspr~para1iomiaiatwe~.Io'Jwers~d

mvariouscondiments seleCted . Figltrett.

'1J1e3VPSEMmipro~I.1s offishanchovies,,~oys~ ~dt9ql~sWdentinepujp,ti&snempJ.iJhQlogiqil changes observedin

,clockwtse

mannerPOStimmersion.

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