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St.John's
EVALUA nON OF PRESERVATlVEMETHODSFORLUMPFISH (Cycioptenalumpus)CAVIAR
By SANDRA WHITEWAY
Athes issubmittedto theschoolof GraduateStudies inpartialfulfilmentof the requirements
forthedegree ofMasterofScience
Departme ntof Biology MemorialUniversityof Newfoundland
September, 1997
Newfoundland
...
ofNauonalCanadaUUrl:IIY g,U'luU"'""lU. . ' .... UU1 ...duCanada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395,rueWellington OttawaON K1A0N4 OtlawaON K1A0N4
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ABST RACT
Lumpfish. caviar was prepared usingvarious preservationtechniquesinclud ing pasteurization. temperature control.and chemical additives.A she lf-lifestudy was conducted on the various caviare samplesduringwhichmic robia l quality and proximate analyseswas examined. The chemical compositionwasfound to be similar to that of othercommercial brands.The microbialqualityindicated that the roe used for production of the finished caviare prod uctmay have beenof poor quality.TheIum pfish.
caviare was foundto be freeofmost food bornepathogens.Thepredominan t bacteria isolatedwas a gram positive cocci(-9 5% ) whichis probablyaStaphylococcus species.
The averagepH afthecaviare was5.9 and the chemicaladditives hadlimited effectivenessat thispH.Pasteurizationwasfound to producea pooraesthet icalproduct whichmaybe anributedtothe raw productquality.Refrigeration was acceptableas a preservationtec hnique for alimi ted period. Co mbinationpreservationtechn iq uesmay offer the best method forextendi ngthe shelf-lifeof caviare whilemaintainingmicrob ia l qualityandchemical stability.
ACKIIIOWLEDGEMENTS
I wouJdlike to expressmy gratitude to my superviso r,Dr.T.Patel,for hispatience, andadviceduring the courseof thisresearchproject. 1 wouldlike tothankMr.Maurice Whiffe n of NorthAtlanticPackagingfor his timeandcooperation.Mr.GeoffreyWhiteway forhis instruction and assistancewiththe chemical analyses.the Marine Instituteof MemorialUniversity ofNewfoundland for use of thei r facilities.Ms. BrendaWheelerof DFO for loan of equipme nt.I would liketo expressmy gratitudetoMs. KathyPenneyof Jacques WhitfordEnvironment andMr.Bevin LeDrew forsupport and patienceduringthis und ertaki ng.
Iwould liketothankmy supervisorycommittee.Dr.J.Pateland Dr.P.Dabinett,for their time.guidance and suggestions throughout thisproj ect.
A specia l thank you toMr.Jeff Free ma n.lRAPproj ectmanager forproviding financ ialsuppo rt underNRCfIRAPwhichmadethisproject a reality.
iii
TABLE OF CONTENTS
ABSTRACT.
ACKNOWLEDGEMENTS.
CHAPTER I INTRODUCTION. 1.1 Caviar. 1.2 Economics of Caviar 1.3 Newfoundland Roe lodustry. 1.4 Roe Processing.
1.4.1 Separation.
1.4.1.1ManualSeparation. 1.4.1.2Mechanical Separation 1.4.2 Draining.
1.4.3 Mixing witbSalt.. 1.4.4 Filling the Barrels 1.4.5 Curing. 1.5 Caviar Processing.
1.5.1 Labelling. Cartoning and Storage 1.6 Pasteurization.
1.7 Microb iology
or
Ca vta r e 1.8 Objectives...iii
.\ ..•1
..
\ ... . .. ... .. ..2 .4 .5 ..6 .6 .7 ...7 .8 ...9 ..9 14 15 16 . .. .\8 CHAPTER 2MATERIALS AND METHODS ..20
2.1 CaviarSamples. ..21
2.2 Media. 22
2.3 Reference Cultures .23
2.4 Sample Analyses. .24
2.4.1 Chemica l Analyses. .24
2.4.1.1pH .24
2Al~Salt. .25
2AIJ~b. ..25
2.4.1.4 Moisture. ...25
Ut~~. . 26
2.4.1.6Protein 27
iv
2.4.1.7Water Activity. . 28
2.4.2 Microbiologica lAnalyses. . 29
2.4.2.1Total AerobicCounts. . . 29
2.4.2.2Total AnaerobicCounts. . .30
2.4.2.3Total ColiformCounts.. . .31
2.4.2.4Fecal ColiformCounts. . .32
2.4.2.5Bacilluscereus . . 33
2.4.2.6Salmo nellaspp. . . 3S
2.4.2.7CoagulasePositiveStaphylococcusaureus. . .36
2.4.2.8Listeria spp.. 37
2.5 Isolationof PredominantOrganism. . 38
2.6 Growtb profilesof CaviareIsolateforVarious Preservative
Methods. . ..38
2.6.1 Temperature.. .39
2.6.2 Salt. . .40
2.6.3 Growtb Pro fileforSodium Benzoate. ...41 2.6.4 Growt bProfileforPotassium Sorbate. ...42 2.6.5 Growtb Profile for Sodium Benzoate/Potassium Sorba te
CombinationPreservatives. . 43
2.7 StatisticalAna lyses 44
...76 CHAPTERJ
RESULTS. 3.1Cbemical Ana lvses 3.2Microbiologics',Analyses.
3.2.1 Preservativesand StorageConditions . 3.2.2 CaviareIsolatePbysical Parameters . 3.2.3 Caviare IsolateGrowth Profiles
3.2.4 Sialislica l Resul bforPreservativesaod SIDrage Conditions.
3.2.5 StatisticalResultsforBacterialIsolatePbysica l Parameters ..
3.2.6 Statistical Results for Cavia reIsolateGrowtbProfiles. 3.2.7 Cavia re Isol at eCharacteri za tion.
CHAPTER 4 DISC USSIO N.
4.1 Cbemica lCompositio n. 4.2 Microbiologica l Qua lity. 4.3 Product Quality .
....46 47 .48 ..49 . ..66 . ...71
81 83 86
.89 .. .89 ..90 .92
. ...94 .9S ...96 .99 .. .100 ...101 ...103 .104 ..104
. IDS
... . . .IDS
. 108
. 110
. 111
.118 4.4 Caviare Isolate. .
4.5Preserv ationMethods. 4.5.1 Refrige ration 4.5.2 Pasteurization. 4.5.3 Chemical Additi ves
4.5.3.1Activity or Prese rvatives.
4.5.3.2Mode of ActionofChem ical Preservatives 4.5.3.3Acids asChemical Preservatives.
4.5.4Benzoic Acid.
4.5.4.1Chem icalProperties. 4.5.4.2Antimicrobial Ac tivity 4.5.5 SorbicAcid.
4.5.5.1 Chemica l Properties. 4.5.5.2AntimicrobialActivity.. 4.5.6 Sodium Chloride. CHAPTE R S
CO NC LUSIO NS ...119
REFERENCE S .. . .. ..122
AppendixA
Chemical Anal yses-Proximat e .129
AppendixB
MicrobialQuality. 144
AppendixC
Post HocTest Results
BoorerrooiAdjustment. ... . ..16 1
vi
Table 1.1:
Table 1.2:
Table1.3 Table3.1:
Table 3.2:
Table3.3:
Table 3.4:
Table3.5:
Table4.1:
Table4.2:
Table4.3:
LIST OFTABL ES
Newfoundlandlumpfish roeslandings 1970 -1987.-.. Lumpfish Roe ProductionNewfoundland Region 1988- 1992
(kilograms). .4
Pasteurization Time and Temperatures .. 16
ProximateAnalyses Results. . 47
ANOVAResultsforAerobic and AnaerobicStandard PlateCounts 79 ANOVAResultsfor Temperatureand Salt Growth Profiles of Bacterial
Isolate. ..82
ANOVAResults for Aerobic andAnaerobicStandard PlateCounts.. 84 BiochemicalCharacterization oftheCaviare Isolate. . 88 Temperature Ranges of Selected Microorganisms. . 97 RequirementsforChemicalPreservatives. _.102 ThepHvalues needed forvariouslevels of undissociated
organic acids. . 107
LIST OFFIG URE S
Figure1.0 Lumpfish RoeCaviar Process . . II
Figure3.1: Aerobic Counts forChe mica lPreservationMethodsand Normal Storage
Conditions.. . .5t
Figure 3.2: AerobicCounts for Temperature Preservation Methods andNo rmal Storage
Conditions. . ..53
Figure 3.3: Aerobic Counts for Chemica lPreservation Methods and AbusiveStorage
Conditions.. . 55
Figure3.4 AerobicCounts forTemperature Preservation Methodsand Abusive Storage
Conditions.. . 57
Figure 3.5: Anaerobic CountsforChemicalPreservationMethodsand Norma lStorage
Conditions.. . 59
Figure 3.6: Anaerobic Counts for Temperature PreservationMethods and Nonnal
Storage Conditions. . .. 61
Figure3.7: Anaerobic Countsfor ChemicalPreservatio nMethodsand Abusive Storage
Conditions.. .63
vii
Figure 3.8:
Figure 3.9 : Figure 3.10 : Figure 3.11:
Figure3.12:
Figure3.13:
Anaerobic CountsforTe m perature PreservationMethodsand Abusive
Storage Conditions. . 65
Temperature Growth Curve for Caviare Isolate. . 68 GrowthCurveforCaviare Isolate inVario us Salt Concentrations 70 Growth Curve for Caviare Isolate in Sodium Benzoate 73 GrowthCurve ofCaviareIsolatein Potass iumSorbete. . 75 Growth Curve forCaviare Isolatein Sodium BenzoateandPotassium
Sorbate Mixture. . .78
viii
CHAPTER I INTRODUcnON 1.1 Caviar
Theterm"caviar(cavi are)"hastraditiona lly been reservedfo rsturgeon eggs,the mainsourceofwhichhasbeenthe Caspi anSea.TheCaspianSeais bometo threesturgeon spec ies(11).eac hproducingdistinctivecaviar.TheBel uga(Husahusa)hasthelargest eggs that aremostlyblack producingthe finest gradeand mostexpens ive of thecaviars.Osetrov a eggs (Acipenser rudiventris )aregreyorgoldenwhereasServrunga (Acipenserstellatus ) yieldsthesmallestgraincaviarwith acoloursimilartothatof Beluga Cavia r.
1.2 EconomicsofCavia r
Supplyshortagesandassociatedhighprices ofsturgeon caviarshasled to the developmentof caviarsubstitutes.ThemostimportantoftheseareSalmon.Whitefishand Lumpfis hCaviars (1t).Cavia rsubstitutesmustindicateon thelabe lthesourceoftheroes (17).Colouri ngis allowedincaviar substitutesonly(4).ThusLumpfishCaviarsubstitutes maybedyedblac kto imitateBelugaCaviar,orredtoimitat e SalmonCaviar(ll,4 1).
Caspian Caviar ofeither RussianorIranian origin accountsfor98 percentofNo rth Ame ricanimportsasof1987.The remai ning2percentwasimported fromChina,Romania,
and/or Czechoslovakia. The ave rage wholesale pricefor impo rtedcaviarin 198 7was S190.00 (US) perpoundfor pasteurizedand vacuwn -pac ked( T able1.I}.Domestic sturgeo n caviarsoldfor half thcpriceof imported at 595.00(US)perpoundLumpfishCaviarhad a wholesale price ofS15.00(US) perpound,Whitefish Caviar wasS16.00(US) perpound andSalmon Caviarwas535.00 (US)per pound.. Thewideprice differences reflectthe different and distinctnichesthat theseproducts occupy in thecaviarmarket(II).
1.3 Newfou ndla ndRoe Ind ust ry
Incepti onofthe lumpfishroe indusny for Newfoundlandbeganin thelate 1960's.It hascontinued togrow(table 1.1,1.2) tosuch an extentthatearningsfrom Iwnpfishfishery may represent asignificantportionof afisherman 'sinco me(11).Newfound landand Labrado rhas becometheworld'sleadi ng produce r andexporte rof bulk saltedroe (I I).
Before 1990,all of value-added con version ofbulk saltedroe tobottledcaviar(11,8)has taken place outs ide Canada.A[ presen t,thereare 3or fewerproducers(8) ofthe caviarin Newfoundl and(Of0designation)Region.
Table1.1: Newfoundlandlumpfishroeslandings1970- 1987,- Landingsin metrictonnes/valuesin$'000.
LumpfishRoe
Year landings Value
1970 21 5
1971 156 33
1972 204 53
1973 153 51
1974 60 23
1975 94 41
1976 320 408
1977 503 261
1978 942 577
1979 930 619
19 80 577 399
19 8 1 846 601
1982 795 565
1983 1,068 770
1984 938 680
1985 1.225 961
1986 2,048 2.997
1987" 3056 11,658
-1970~1984figures:DepartmentofFisheriesandOceans,StatisticsBranch.Nfld Region
1985·1987figures:DepartmentofFisheriesandOceans,StatisticsBranch, Nfldand Gulfregions
bfiguresarepreliminaryandonlyincludelanding as of October 20,1987.
Source;(10)
Table 1.2: Lumpfis h Roe Production NewfoundlandRegion198 8-1992 (kilograms ).
Year Quantity Prod uct Form 1988 1.703,388 Brinecured lwnpfish roe 1989 \.940.903 BrinecuredIwnpfishroe
\990 953,486 Brine cured Iwnpfishroe&caviar
\991 \.629.616 Brine cured lum pfish roe&caviar 1992" \.619.025 Brinecured lumpfish roe.caviar,
canned caviar and frozenroe.
':preliminaryandsubjected torevision Source:(6)
1,4 Roe Processing
Ca viaryield and qualityare dependantupon thefishhandlin gpractices(harvestto roe extraction).fishing season.fishsize.fish species. areaofcatch and maturity(10, 14).
Thelumpfishgillnet fisherie soccurs duringthespawning period(mid-Aprilon).Generally.
netsof 10-11inch mesh sizeareused in random,gangsor fleets (10). Malelumpfishare smaller than thefemale sandcan usuallypassthrough themesh. Should themales become
enmes hed, theyare easilydistinguishedfromthefemalesbytheirredbelliesand are returnedto thewater.
Roe isremovedimm ediatelyafter catch.Theroeisremovedbycuttingopenthe belly ofthe femaleandcarefully extractingthe twosacsof eggs.It isim portantduri ngthe extra ctio nprocedurethattheroesacsremainintactandclean(II).Ifthe sacsareremoved intact, the roeremainsrelative ly sterile.Gut fluidsarenot perminedintheroehol ding containerssincethisfluid will introducecontaminatio n. The remov edroe isplacedin containers whichmeet fishinspectio n regulatio ns.Theroeisiced. Care mustbe taken to ensurethat asufficie nt ice-to-roeratio isachieved(10).Roemust be processedassoonas possib le.Genera lly, insufficienticeisused tochillroepriorto arrivalat the plant,thus reinforcing theneedtoproc essthe roe immediately.
Upon receiptat plant,theroe is drained byplacingapproximate ly2Jkgofroeona I-I.Smmmesh sizescree n.Itisweighed. Roeshould not be keptlongerthan overn ight priortoprocessing (10).
1.4.1 Separation
Thefirststep in lumpfishroe proce ssin g is the separatio n of the eggsfromthesac.
Thiscanbepreformed eith e r manually or mechani cally(10 ).
1.4.1.1ManualSeparation
The manualmethod of separationuses stainless steel screens set ina stainlesssteel frame.Itisrecommended thatthree screens of 10mm mesh, 5 mmmesh and 3 nun mesh be used to produce acleaner roe containingaminimal amountof blood, gut and sac (extraneous)material (10).
Theroe is spreadonthe topscreenandgentlybut firmly rubbed acrossthe screen using the palmofthe hand Eggs sepa ratefrom the sacand rail throughto the second screen.Extraneous materialmay also pass throughtothe secondscreen.The remaining extraneous material willbescreened outas the eggs pass throughthe secondand third screens. Screens must be cleanedfrequently to protect roequalityand limit contamination.
Eggs fall to a draining screenandare coveredwith aplasticsheetto limit contaminationas they draintlu).
1.4. 1.2 MecbanicalSeparation
Themechanical separation method is preformedon a separatingmachine.The machine containsa stainlesssteeldrumwhichhasrows of 5mmperforations.There are two to sixpaddles whichrotateinsidethedrumand push theeggs through theperforations.
EggsfaU ontoa2 mmmesh drainingscreen. while the extraneousmaterial rema in inside
the drum.The dnun is cleaned frequentlyto protectroe quality and limit contaminatio n (10).
1.4.2 Drain ing
Eggs are left on the draining screenfor severalhoursto remove as much water as possible. Duringthis time the drainingscreen is covered to protect the eggs from contaminat ion.Eggs must not be piled more than 25 em deep to ensure the bottom eggs are not damaged from pressure.Placingthe draining screens on an incline speedsthe draining process.Thelength oftimethe roeis drained will influencethe amount of roe needed for topping-upprior to shipping.Thebetterdrained (longer the draining time) the roe is,the less roe that isneeded fortopping- up( 10).
1.4.3 Mixin gwith Salt
After the roe is drained, the roeis mixed with finefisherysalt whichacts as a preservative.The amount of salt used( 12-20%) is conside redborderlineforthepreservation purposes and thus theprodu c t must be kept chilledduring and aftercuring to maintain productquality (10)..
The proper mixing ofthe salt androe is essentialtothefinalproductqua lity.Mixin g ispreformed in three,fouror fivebatchesper barrelto ensurean evendistri butio nof salt and roe.Roe andsalt for each batch are separate lyweighed and mixedby hand. Thesalt-to- roe ratio isdeterm inedbybuyer spec ificationsandis usual lybetween 12-20%.A minimwn ofgreate r than9% salt isnecessarytopreventspoilage oftheproductfrom microorganism s suc hasClostridium botulini um andothe rhalo-toleran t bact eria(10).
1.4.4 Fillingthe Barrels
Batches ofthe roe-sa ltmixtureareplacedinherringbarrels.Barrelsare filled. rimsclean ed, covered and sea led.Barre ls are storedinachill roomat34°C.Barre ls shouldbele ftup rightso theroe can settle and the following daythe barre lsshouldbetopped-u p wit hsalted roe prep ared thesam e day asthosein the barrels. This is nec essarybecaus eafte r 24 hou rs the contentsofthebarrel willshrink!settleand occupyapproximately3.4of the volwne of thebarr e l. Thelid is replace d andthe barrelisfilledwith a brinesolution throu gh the bung hole toexpel any air present.Barrel s shouldbeleftuprightand top ped-upnext day toavoid probl em s.Airwillreactwithnaturaloils inthe roe andcause rancidity which is oneof the majo r problem swithlumpfishroe(10).
1.4.5 Curing
The barrels arelefttocure for 12-14days.duringwhic h they are rolled and topped-upwith brine.Rolling the barrels hel ps to mixroe,saltandbrine thereby preventi ngspoilageof me roe.Barr e lsorcuredroe are kept chilledand during the firstmonth ofstorage arerolledand topped-upwith brine oncea week. Thisprocessisthenrepeatedonceamonth for the remai ningstorage period (10).
1.5 Caviar Processing
Theproductionofcaviar fromcuredroe requiresaseriesofproceduresthatincl ude salt reduction, roe cclourauon,pHadjustment, add itive incorpo rat io n. packaging, pasteuri za tion (ifdesired ),labelling and storage.Aflowdiagram outliningtheprocess is presen tedinFigure1.The firststepinthe caviarprocess is todetermineifthe barrelledroe issuitableforprocessing. The following conditions mustbemelfor the cured roe tobe suitableforcaviar processing:
Thetem pera ture of thecuredroeshouldbe00- 3°e.
Notrace of rancidoroffodours.
Theroemustbefreeof objectio nal matter suchasblood, liverorsac material.
Theremustbeno foreignmaterialpresen t inthe curedroe.
Figure legend for Figure 1.0.
Flow diagram of the lumpfishroe caviare processillustrating critical stepsinthe manufacturingprocess.
Source: Departmentof Fishe ries.GovernmentofNewfoundlandand Labrado r.1989.
Industry SupportServices ReportNo. 43.
\0
Preparationof Drainingof Barrelled~ Solution;Salt
Roe .--~ Reduction.Dye
addition.pH Adjust
Sepa~~~u~:nFrom ~RoeAdded to Solutio n
Drain Roe Overnight ~Jarand Lid Preparat ion
Vacuum SealJars ~
figure1,0 LumpfishRoe Caviar Process 11
Fill Jars
Label Jars
The cured roe must be checked for the percentage of broken eggs.
The salt content mustbechecked to determine theproper ratio of dilution for the desalting process.
The pH mustbemeasured to determine the amount of acidulentnecessary for pH adjustment (10).
After a barrel of cured roe has been found acceptable for caviar processing it must bedrained A screen is clamped onin place of the tid The barrel is inclined to allowexcess brineto drain.This takes approximately30 minutes.During this time.the results ofthesalt and pH content areused to calculatethe volume of water. dyeand acidulent necessary to adjust these factors to the desired level (10).The salt content is adjusted using the following formula:
wt.of me (kg)x0.90x salt content-desired salt content =Water(L) Desiredsalt content
Thisformula determines the amount of water in Iitres thatis needed to dilutethe roe.Dye isadded (10).
The amount of acidlacidulentrequired to adjust the pH is determined by trial and error.
The amount required will depend upon the pH of the dilution water.pH of cured me and the ratio of roe to dilution solution.Citric acid is used as a acidulent and the pH is monitored until the desired pHlevel has been attained (10).
12
The dilution solution ispoured into a largevatand the roeisadded.. Thetem perature must be maintainedbelow15"C.Tberoeremains inthe dilution solution for 30-45minutes dependi ngupon thedye used. This isnecessaryto elimi na tethepossibilityofinco nsistent dyeing(lO).
Theroeandsolutio nare slowlyagita tedtoensure properandthoroughmixing.Sac material,bro ken eggsandotherde bris will floatandareskim medof(I0).Theroe isnow dyed, salt reducedand acidadjustedThe roe ispouredonto drainscreens which are capab le ofholdingup to25kgof caviarmaterial. A portionofthedilutio nsolutioniscollectedand re servedfor rinsingout thevats.The screenswiththedrainingroe are plac ed ina chill area on racks which areincli ned.at a20eangleand do notallowdrainingfrom toptrays tofall on lo wer trays.The roeshould beleftovern ightto reduce the amount offreeliquid(10 ).
Roeis removedform the chill area after draininghasbeencompleted. At thistime desiredaddi tivesare added.to thecaviar.Com mo n additivesare oil (whic hincreases the product"flow". increasesthe jar filling capacityand givestheproductaglossy appearance).
antioxidan ts.flavourenhan cers. spices.sugar,and/or preserva tives.Theadditives used are often related.tomarket requirem en tsandoppo rtun ities(10).
Jars are cleanedbyremo ving any debris.washed anddry.Lidsare usuallypackaged under sanitarycondit ion sand do not need.anyprewash ing. Jars arefedto the filling
13
machine and lids are fed to thevacuum closingmachine(10). Jars are usuallyfilledby eithe r an automatedorsemi-auto mated filler.Caviarfillersfill from thebottom of the jar totherim. Themethodsof cappingarescrew-e nscrew -off and crimp-enscrew-off Lum pfishcaviar are most commo nlyvacuumsealed byeitheramechanicalvacuum.steam evacuationor hermetical selfsealing caps.Closedjarsshou ldbecod edonclosi ng or imm edi atelyafter(10).
Closed jars periodic allyundergoaqualitycontrolcheck forweightand vacuumintegrity.
Thenum ber of jarswhichare chec kedshou ldbepredeterm ined accord ing toastatistica l sampli ng plan. Jarswhichwe igh greater orlessthanestablishe d tolerancelimitsare rejected. Itmaybenecessarytoreadjust the filling machinetoensure the appro priate volumeisdispensed.Similar ly.ifthereis a problem with thevacuumsealedlids.the vac uumsealingmethod must bechecked, theproblem identi fiedandcorrect ed(10).Atthis stage thecaviar is ready tobelabelled, cartoned andstored orif des iredthecaviarwill unde rgopasteurization.
loS.. Labelling,Cartoning andStorage
Labels forcaviarsoldinglasscontainersare generallyappliedtothelids.Thelabell ing step usua llyoccurs afterthejars are cleanedanddried.Lumpfi sh caviaris usually packa ged as 6 or 12 jars to a case.Oncejarsarepacked incartons thefinal product isheldinstorage
14
priorto shipping. Lumpfishcaviar shouldbe stored,shipped and handledatchill temperatures (2c- 4CC ) at alltimes after processing (10).
1.6Pasteu rizatioD
Pasteurizationof cappedcaviar jars is done in eitherbatchesorcontinuously.Batch pasteurization requires that closedjars are collected in a basketand immersed into a temperature regulated bath.Continuouspasteurization req uiresthatthe closedjersbeplaced ona conveyor belt that passes througha pasteurizationtunnel(10).
Regardlessof the pasteurizationmethod. the paste urization process is followed by a coolingunitwhichuses lukewarm waterto avoid cold shockcracking of the glass (10).
Pasteurizationtemperaturesandtimes generally used withinthe industry are presentedin Table1.3.Pasteurization dependson the come-uplime. This is the time required to raise thetemperature of the produc t at the centreof the jar to pasteurizationtemperature.The exact time and temperature dependsuponthe shape and dimensions of the container,the initialbacterial loadand the ambienttemperature (10).
15
Table 1.3 Pasteurization Time andTemperatures
Jar Size (oz) Time(min) Temperature(oC) Internal Temperature(oC)
2oz " 23 73.9° 65.5°
4oz" 41 73.9" 65.50
7oz · 53 n.9" 65.50
Iaoz" 91 73.9° 65.5°
50 0z•• 120 57"
toO oz••• 36 75_80° 750
50 oz'"" 60 710
Source:RomanoffFoodlnc.
Source:Dr.Iredale.Freshwate rInstitute.WinnipegManitoba Source;Dewar.LiptonandMack,197 1
TableSource(to ).
1.7Micr obiology orCaviare
Caviare may be preserved chemically.orthrough the useof temperaturepreserva tion methodssuch as refrigerationor paste urizat ion.These preservativemethods may resultin thereduct ionofbacterial loads.retard or inhibitmicroo rganismgrowth thereby extending product shelflifeandensuri ngprod uctsafety.Theintrins ic parameters (pH.salt.moisture.
wateractivity.etc.)of many food productsincludi ngcaviare are suchthatbacteri algrowth anddeco mposit ionare not retarded andconsequently neitherare microorganisms responsiblefor foodinfections andintoxicants.Themethod of preservationchose nwill
16
dependgreatly uponthetargetshelflife,theinte nded consumer,prod uctuse, storage, shippingrequireme nts, importcountryregula tion s,buyerspecifications andthe expected initial bacterialloads.
Theava ilability of published litera tureoncaviare microbiologyis sparse (particularly whencomparedto othe rfood products), dated and oftenfoundin obscure sources. A majority ofthepublished materialon caviareis basedon Soviet research. doesnot deal with lumpfish caviare per sayandis based onmethodo logieswhicharenot necessarily comparableto North Americanstandard meth ods.Informa tionon microbialand chemical qualityof caviareheld by commercial producerswouldbeconfidentialand therefore unavailab leto thepublic.Inaddition.concerns have been raisedbygovernme ntagencies withrespect to the lumpfish caviare prod uctsafety,andas suchthere is anide ntifable data gap.
Totalcoliforms.fecal ccliforms,Eshenchia coli,Staphylococcus aureus, Salmonella spp.,Listeria monocytogen es,andBaci llus cereus are known causativeagents or indicators of food intoxicationsorinfections.Thisstudy willundertaketo determ ine the occurrence of the microorganismsinlumpfish caviare. The total aerobic and anaerobiccontentof lumpfish caviare willalsobeexaminedas anindica torof productquality,preservative effectiveness and shelflife stability.
17
1.8 Objectives
Caviar presentlyproduced in Newfoundlandusessodiwn benzoate(40)forpreservation.
The EEC(15), Korea and othercountries (40) prohibittheuse of sodium benzoateasa preservativein caviar.The use of sodiumbenzoate in combination with potassiumsorbate up to 400 ppm would be acceptablefor mostEECcountriesundernewguidelines.
Therefore,tomarket caviar in thesecountries.preservationmethodssuch aspasteurization, refrigerationortheuse of acceptablechemical additivesmustbeimplemented.
The proposed studywillinvestigate:
Alternatepreservation methods for caviare.
., Determine if alternate preservation methodswouldensureacceptable microbiologicaland chem icalquality.
3. Determinethe most effectivealternativepreservationmethod.
This study willexaminethefollowing preservat ionmethods:
Sodium benzoate.
2. Mixtureofsodiwn benzoate(100 mgtkg)and potassiumsorbate(300mg/kg).
3. Mixture of sodiwn benzoate(200mg/kg) and potassium sorbate(200 mgfkg).
4. Mixture ofsodiwn benzoate(300mgfkg)and potassiumsorbate( I00 mglkg).
5. Refrigeration(4°C).
18
6. Pasteurization(55°Cfor135minutes).
7. Pasteurization(70°Cfor 45minutes).
19
CBAP TE R 2 MA TERlALSAND MET H OD S
NorthAtlantic Packaging is a secondary processor of lumpfishroecaviar basedin Newfoundland. producing50 and 100gramproductinglass jarswithsealed metal caps.
The productsan: preservedwithbrine and sodiumbenzoate(0.0 8% ).Theproductshavethe requirem ent forrefrige ratio n uponopening.Theprod ucts are retailedat a variety of outlets inCanada(40 ).
Presentmarketsare limitedto theCanadian retailmarket. A new potentialmarket in South Koreadoes not pennit the use ofsodiumbenzoate.The SouthKoreans requireall natural ingredientsto be used(40). North AtlanticPackaging produced severalexperimental runs of lumpfishroe caviartoexamine alternativepreservativemethods.determine ifthe alternativemethodswouldensuremicrobio logica landchemical quality,anddeterminethe most effectivealternative method.Thealternativemethods examinedwere :
I. Pasteurizatio n55°C.
2. Pasteurization70°C.
3. Refrigeration.
4, Combination of sodium benzoate/potassiumsorbate(3:l).
5.Combinationof sodium benzoate/potassiumsorbate(I:3).
6, Combinationof sodiumbenzoate/potassiumsorbate(I;I).
20
The present preservativemeth od using sodiumbenzoate (80mgtkg ) was also examined.
Alternative methods 5 and 6 would be applicableformost EEC countries underproposed new guideli nes(15).
2.1 Cavia rSamples
Caviare sampleswere obtained from North Atlantic Packaging contai ningsodium benzoateat 80 mgtkg.NorthAtlantic Packaging produced specialruns to make the caviare sampleswith the preservativelevel s at:
I. Mixtureofsodiwnbenzoate(100mglkg) and potassium sorbate(300 mg/kg).
2. Mixture of sodiumbenzoate (200mglk g) and potassium sorbate (200 mglkg).
3. Mixture of sodiumbenzoate(300mglkg) and potassium sorbate (100 mglkg).
4. Uri-preservedcaviare.
Caviar samples speciallyprepared for this study were collected from North Atlantic Packaging immediatelyafterproc essing. The microbiologica lsurveywas initiatedwithin three hours of processing.Samplesthat werechemically preserved or pasteurized were stored at 20G
e
(holdingtem pe ra ture ) and 37°C (abusive temperature) for the dura tion of the study.Samp lespreparedwithnoprese rvativefor refrigerationwere storedin arefrigerator at 4GC(ho ldingtemperature)and 20GC(abusive temperature)for theduration of thestudy.21
Commercial samples were obtaineddirectly from the processi ngline and were stored at 20°C(holding te mperature) and 37°C (abusive temperature).
Samplesforpasteurizationcontainednopreservati ves and wereobtaineddirectlyfrom theprocess ing line.They were transportedto thelab where theywereplac ed in prepa red wate rbathsof70GCand55GC.The pasteurizationprocess startedwithin15 minu tesafter removalfrom theproce ssing line.Sampleswere pasteurizedforSSminute s at70°Cand 135 min utesat 55°C.Pasteuri zedsampleswerestored at 20GCand 37G Cfor the duration of thestu dy.
2.2Media
All mediausedin thisproject were ofreagent orlabo ratory grade.Chemicalsandmedia were obtainedfromFisher ScientificLimi ted, Dartmouth,Nova Scotia orSOHChem ica ls, Dartmouth,NovaScotia.Potassium so rbate andsodium benzoat e were offoodgrade quality andwere providedby NorthAtlan tic Packaging.
The following mediaand chemicalswere obtainedfrom FisherScientific Limited.
Dartmouth.NovaScotia: Listeriaenrichmentbroth;UVM listeria enrichmentbroth;Oxford liste ria selectiveagarandsup pleme nt;Typticase soy agar; coagulase plasmawithEDTA;
BairdParker agar,nutrient broth;tetrnthionate broth;brilliantgreendye ;potassiumiodide;
22
iodine;bismuthsulfiteagar;brilliant green sulfaagar, XLD agar; Hektoen Entericagar;Egg yolk telluriteem uls ion;egg yolk50% emulsion;purplebroth base;motilitytest mediwn;
levine'sEMB agar, Gramsta in test kit;trypncasesoybroth;peptone;nutrientagar.tryptone bileagar, 0.45micro n85 mm cellulosicmembrane filtersand yeastextra ct.
Thefollowing chemicalsand media wereobtained from SDHChe micals.Dartm o uth, NovaScotia: PALCAMlisteria selective agar and supp lement;sodium chloride; vio letred bile agar;stan dar d methodsagar;SeleniteCysteine; Bacilluscereus agar, AnaerocultA and Anaerctest.
2.3Reference Cultures
Listeria cultures were obtainedfrom Dr. T. Patel. Memo rial University of Newfoundland, St.John's.NF.TheListeria culturesused wereLister ia monocy iogene slhb (HPB #395),L.lnnocua (I-iPB#8)andL Ivanovt i(HPB#28).TheBacillus cereus(E 14579 ), Salmo ne llatyphimurium(ATCC 14028), Escher ichiacoli (11775 ),Clostridium sporoge nes(19404) andStaphylococcusaureus(E 12600)cultureswere obtai ned from the Department of Biolo gy.Memorial Universityculturestoc k collection.
23
2.4Sample Analyses
Caviare sampleswere exam inedwithin2ho ursof processing.and after2.4.8 and 16 weeksof sto rageof samp lesatnonnal(20°C ,4°C)andabusive (37°C.20°C)conditions.
Sa mpleswere examinedforchemica l and microb iologicalquality.
2.4.1 Chemica l Analyses
Proximate(chemical)analyseswere preformedas perstandardprotoco lsestablishedby theAssociatio nofOfficia lAnalyticalChemists(I). The chem icalqualitywasdete nn ined byexaminationof caviaresamples for pH. salt. moi sture . fat,pro tein. and water activity.
2.4.1.1pH
Ten grams of caviarwere homogenizedwith 90mlofdistilledwa ter.ThepH was read witha standar dize d pHmeter(Orion.FisherScientific Ltd.). The probe wasimmersed in thesamples and the digitalreadings recorded (I).
24
2.4.1.2 Sa lt
Ten grams ofcaviar were homogenizedwith90mlof distilledwater(same oneused for pH determi natio n). Salt analyseswe reconducted usinga SMIOsaltmeter(Presto-Tek Corporation)standardiz edwithare feren ce saltsolution.The probewasimmersedin the samples and theanalo g readingsrecord ed(I).
2.4.1.3Ash
Twogramsofcaviar wasplaced in anasbingcruci bleand weighed. Thecruc iblewas placed in a muffle furnace at 525G
e
for24hours.The cruciblewasre-weighedandthe ash contentcalculated(I).2.4.1.4Moisture
Two grams ofcaviarwasplaced inanalum inumplanchet andweighed. The planchet wasplacedina forcedairconvectio noven atlODGeuntilconsistentweights wereobtain ed.
The sampleswere coo ledina desiccat orbetween weighing..Thelossinweightisreported asmoisture loss (I).
25
2.4.1.5 Fa t
Tota lLipid(fat)conte nts(I)were determ inedby the soxhlet method (36)usingthe teeatorsoxhletunit.Threegramsof driedsamplewasplacedinto thethimble.Thethimbles were attachedto adapters andfatfreecononplugs wereplacedon top ofthesample.The thimbleswereinsertedintothecondenser.The condenserknobswere intherinse position.
Theknobs weresettotheboiling positionsothatmagn et fastened tothethimbleadapter.
Theknobwasadjusted totherinse position.Extraction cups of known weightcontai ning bo ilingchips and25-50 ml ofhexan e wereplacedinto thecondenser.Thehandlewas lowereduntil thesafety catchengage d(36).
The extractionknobwas settotheboilingpositio n.Thethimbles wereimmersedinto thehexane solven t,Thesamplewasboiledfor I hourand rinsed for 2hours.Thecondenser valvesare closed after therinsingcycle is completedby turningaquartertum. Upon collectio n of
me
remaining solventinthe condenser.the-AIR" button on theservice unit waspressedandthe-EVAPORATION" valve ontheextraction unitwas opened. The"EVAPORAnON""valvewasclosed,extraction cupsrelc:asedand removed.Thecupswere
placed inan ovenat80°Cfor20minutes.The thimbleswereremovedfromthecondenser usinga thimb leholder.The instrum ent wassh ut down (36).
26
The fat content was calculated as follows:
Fat%=
2.4.1.6 Pro tei n
100xLipid Weight Sample Weight
The crude proteins (%N x 6.25)were determined( I) by the macro-Kjeldahlmethod(35).
Atecator digester and distillationunit were employedto preform the macro-Kjetdahl(3 5).
A 2.5gram samplewasplacedin the digestionflas k.Added to theflas k sequentlywere 15 g NazSOhIg CuS04•one or two selenize dboilinggranulesand 25m.Lof concentrated H2S04, Themixturewasdigesteduntil the solutionwas colourlessor alight green (approximatel y 2hours forinorganicmaterial).The samplewascooled for an additional 30 minutes. Two hundredmL of waterwascautiouslyaddedto the cooled sample. Additiona lboilinggranules (if necessary) were added to prevent bumping(35).
Onehundr edmL of0.1N HCIwerepipetted into a 500mL Erlenmeyerflask.ImLof Conways indica torwasadded. Theflask was placedunder the condense rensuring that the condensertipwasimmersedin the acidsolution.The Kjeldah lflask containingthe digested samplewastiltedand 100 mL of 50% NaOH solutionwasadde dwithout agitation.The
27
flaskwas immediately connected to the distilling bulb of thedistillation apparatus.The flaskwasrotated to thoroughly mix contents (35).
The samplewas heated until all ammonia had passed over the standard acid.
Approximately[50mLwascollected and removed immediately. The tip ofcondenserwas washed and excess standard Hel in distillatewas titrated with NaOH standard solution (35).
The precenr nitrogen(we t weight basis) was calculated as follows:
%Nitrogen(wet)= (A-B)x I 4007 Weight(g) of sample
where A=volume(mL)standard HCI x nonnality of standardHe!.
B=volume(mL)standard NaOH x normality of standard NaOH.
2.4.1.7 WaterActivity
Samplesof caviar were placed in the water activity (I) containers.The containers were placed in the CX-l Decagon water activity unit (Decagon Devices Inc.). Efficiency of the water activity unitwas verifiedwith a KNO)solution which has a water activity of 0.936.
28
2.4.2 Microbiological Analyses
Microbiol ogicalanalyses werepreform edasper standard protoc ols establishedby eithe r Heal th ProtectionBranch(HPB) of Healthand WelfareCanadaorFood and DrugAgency (FDA)oftheUnitedStates.The microbi ol ogicalquality was determinedby the examinatio n of caviaresamplesfortotalaerobiccounts.total anaerobiccounts,totalcoliformcounts, fecal coli formcounts(E.coli),Bacillus cereus,Salmonella spp.,coagulase positive Staphylococcus aureus andListeria spp.
2.4 .2.1TotalAerobicCounts
The totalaerobiccountwascondu ctedas per HPB standardmethod MFHPB- 18 (18)and theprotocols outlined bytheUSFDA(37 ).Approxima te ly11.0g samples of caviar were sto machedin the Stomac he rLabBlender(Canlab Divisio n,BaxterCorporation,Moun t Pearl,NF) for I minu te with 99 mlof0.1%sterilepeptonewa ter.Decimaldilutionswere prepar edfromthe 10.1dilution(usua lly upto 10-6dilutio n) bytransferringI.lmlof the previo usdilutionint o 9.9ml of0.1%ste rile peptone waterina testtube (18,37).
Eachdilutionwas agitated to resuspendmateri al priorto plating.One mlor
o.
Imlof therequireddilutionswere pipettedinto approp riatelabelledduplicatepetridishes.Twelve (12)to fifteen(18) ml oftem pered standar d methods (platecount)agar were poure dinto29
eachplate. The platesweremixedbyrotatingandtilting. The plateswereallo wed to solidify.Plateswereincubatedintheinverted positionat 30°Cfor48:l:2hours.Colonies ontheplates were enumerated usingaquebeccolony counter (Fishe r Scien tificLtd.
Dartmouth,NS).Total aerobic countsweredeterminedusingenumerationguidelinesasper USFDA (1984)standard meth ods(18,37).
2.4.2.2Total AnlerobicCounts
Approximately11.0g samples ofcaviarwere stomac hedintheStomac he rLab Blender (Canlab Division,Baxter Corpora tion, Mount Pearl,NF)forI minutewith 99mlof0.1% sterilepepto ne water.Decima ldilutions werepreparedfromthe 10-1dilutio n(usua lly up
(0
Icr
diluti o n)bytransferring1.1mloflhe previo us dilutio n into 9.9 ml of 0.1%sterile peptone waterin a test rube.One mt or 0.1ml of the required dilutionswere pipetted intoappro pria te labelled duplicatepetridishes.Twelve (12)to fifteen (15) ml oftem pered trypticase soy agar were pouredinto eachplate.The platesweremixed by rotatingand tiltin g.Theplates were allowed tosolidify.Plates wereplaced invertedinto an anaerobe jar withAnaeroc ultA gas packageand ananaerobe condition indicator (Anaerotest).The anaerobe jarswereincubated at 35QCfor 48±2hours.Colonies on the plateswere enume rated usinga Quebec colony
30
counter(FisherScientificLtd. Dartmouth. NS).Tow anaerobicvegetative counts were determ ined usingenumeratio nguidel inesas per USFDA (1984)standard methods (37),
2.4.2J TotalColiformCounts
Thetota lcolifonnanalysiswasconductedas per protocolsoutlinedinHPB MFLP-43 (18). Approximately11.0 g samplesof caviar were stomachedin the StomacherLab Blen der(Canla bDivision,Baxter Corporatio n, Mount Pearl,NF)forI minute with 99ml of0.1%ste rilepeptonewater.Decimal dilutio ns werepreparedfrom the10'1dilution (us uallyupto 100{; dilution)bytransferring1.1 mlofthepreviousdilutioninto 9.9 mlof0.1
%sterile peptone water ina test tube (18).
Eachdilution was agitated to resuspend mate rialpriorto plati ng.One ml or O.I ml of therequired dilutions were pipened intoappropriate labelled duplicatepetri dishes. Twelve (12) to fifteen(I S)mlof tempered violetredbile agar werepouredinto each plale.The plateswere mixedbyrotatingandtilting. The plateswere allowed to solidify.Plates were inc ubatedintheinvertedpositionat37°Cfor 48±2hours(I8).Colonies00theplateswere enumeratedusing a quebec colony counter(FisherScientificLtd, Dartmouth.NS).Total coliform counts we redeterminedasper guidelinesusedfor totalaerobiccounts(18,37).
3\
A direct plating method forthe determination oftotal colifonnswasused instead of the standard Most ProbableNum ber(MPN) method becauseofspace andequipment consid erations.Themultiple tube methodrequiresfifteen(15)tubesper dilutionfor each sampletobe inc ubated in a colifonnwaterbath.Thiswould haverequired more waterbaths than wereavailable ,therefore a directplating method was theonly viableoption.
2.4.2.4 Fecal ColiformCounts
Enum eratio nofFecalColifo nns(E. coli)wasco nducted asper protocolsoutlinedin HPS MFHPB-27methodolo gy (18).A direct platingmethod forthedetermina tionoffecal coli form(Esherichiacoli)wasused instead of thestan dard multipletube method because ofspa ce andeq uip ment considera tion s.
Approxi mate ly 11.0 gsamplesofcaviar weresto mac hed intheStomacher Lab Blender (Can lab Division,Baxter Corpo ration.,Mount Pearl,NF)for1minute with99mlof 0.1% sterilepeptonewater. Decimal dilutions were prepared from the 10'[dilution(usuallyup to 10-6dilution)bytransferring1.1 ml ofthepreviousdilutioninto 9.9 ml of 0.1%sterile peptone water in a testtube (18).
Each dilutionwasagitatedto resuspendmaterialpriortoplating.In duplicate, 0.5ml oftwoco nsec uti ve decimaldilutionswere plated onamembranefilter overlayingnutrient
32
agar.Theinnoc ulumwas spreadover the membrane filterwitha glass spreader. Carewas takento spread the innoculumevenlywithoutspillingit overthe edge ofthe membrane filter.Aftertheinnoculumwas absorbed. the plateswere incubatedright sideup at37°C for4hours (18).
Themembranefilters were removedwithsteri leforceps aftertheincub ation period of four hours.The filters were transferred to preparedand airdried tryptonebile agar plates.
Theplateswereincubatedupright at 44.SoCfor18·24hours (18).
After incubation at44.s°C,the petri dishcovers were removedand wipeddryand2.0 ml ofindoJereagentplacedin each cover.The membrane filter was lifted and placedin its respective cover so thatthe entireundersurfaceissoakedwiththe reagent.The membrane filter and reagentareleft for 20 minutesatroomtemperature.Removetheme mbrane filter by draggingitacrossthelip of thecover[0remove excessindo lereagent.Drythe filters undera germicidal UVlampfor 20 minutes.The pinkto red coloniesappearing on the membranefiltersare indoleproducersandare enumeratedasE.colibiotypeJ(18).
2.4.2.5Bacillus cereus
EnumerationforBacill us cere uswas conductedas per protocolsoutlinedby the USFDA (37).The USFDA methodwas usedinstead ofme HPB method as a resultof the inability
33
to obtain the base media due to back orders. Both methods aresimilar and either will identify and enumerateB.cereus(37).
Approximately It.O g samplesofcaviar werestomached in the StomacherLabBlender (Canlab Division,Baxter Corporation., Mount Pearl.NF)forI minute with99 mlof 0.1% sterile peptone water.Decimaldilutionswereprepared from the IO-ldilution (usuallyup to 10-0dilution)bytransferring1.1ml of the previousdilution into9.9ml of0.1%sterile peptone water in a testtube(I8.37)_
Duplicate Mannitol-EggYolk-Polymyxin (MYP) platesper dilutionwere inoculated with 0.1mleve nly distributedoverthesurface with a sterileglassspreading rod.Plates wereincubated invertedat 30°Cfor 24 noUTS. Plateswere checked for typicalB.cereus colonies(pink colour with precipitatezone indicatinglecithinase production ).Plateswith unclear reactionsor no growth wereincubated an additional24 hours(37).
Theywere no colonies indicativeofB.cereus on the plates.thusB.cereus were consideredabsent. Therefore. itwasnot necessarytoconductconfirmation and differentiationanalysesforB.cereus.
34
2.4.2.6Salmonellaspp.
Isolat ionandident ificatio nofSalmonellawascondu cte dasper HPB MFHPB-20 protocols (18).Approxi mately 11.0g samples ofcaviar werestomac hed in the Stomac her LabBlender(Canle b Division,Baxt er Corpo ration.Mount Pearl. NF)forI minute with99 mlofnutrientbrothas apre-enrichment.The brothwasinc ubatedat35°Cfor 18-24ho urs . One(I)mlofthe incubatedpre-enrichm entbrothwastransferredto9 ml of selenite cyste ine andtetrathion ate broths.The selenitecysteinewasincubate dat35°Cand the tetrathicnate brothat 43°C for24 hours(18).
A loop from eachofthese lectiveenric hment broths werestreaked ontobismuth sulfite agar,brill iant greensulfaagar, XLOaga r,and hektoen entericagar. All plateswere incubated at35°C for24 hours.Plateswereexaminedfor coloniestypica l ofSalmonella (18).
Therewere no coloniesindicativeofSalmonellaon the plates,thusbacteria ofthe genus Salmonella we re conside redabsent.There fore.itwasnotnecessarytoconductbiochem ical screening andserologica lide ntifica tion.
35
2.4.2.7Coagulase Positi veStaphylococc us aureus
Enwneration of coagulase positiveStaphylococcus aureus wasconducted as per protoco ls outlinedbythe USFDA(37 ).Approxi mately11.0 g samples of caviar were stomachedin the Stomacher Lab Blende r(CanlabDivision,BaxterCorporation,Mount Pearl. NF) for Iminutewith 99 mlof 0.1%sterile peptone water.Dedmal dilutionswere prepared from the10-1dilution(usuallyup to 10-6dilution)by transferring1.1 ml of the previousdilutioninto 9.9ml of0.1%sterilepeptone waterina test tube(18,37).
For eachdilutionplated., 1.0 ml (0.3.0.3 and 0.4 ml)ofsamplewasdistributed equally over3 Baird-Parkereggyolk tellurite plates.Innoc ulumwasspread overthe surface of the platesusingasteri le bentglass rod.Plateswere retainedupright untilinnoculumwas absorbed (a pproxi mately10 minutes). Plateswereincubated inverted at3SGCfor45-48 hours(37).
Colonies typicalofS.uureus(circ ular.smooth. moist.convex,2-3mm,gray tojetblack, and frequently having aouter clear zone) were transferredto tubes containing 0.2-0.3mlof brainheartinfusion.The brain heart infusion tubes were incubatedat 3S"C for 18-24hours.
Reconstituted coagulase plasmawith EDTA(0.5ml)wasaddedtothe tubes.The tubes werere-incubatedat 35G Cfor6 hours and periodicallyexamined forclotfonnation.Only firmand complete clots whichstay inplac eupon tiltingwereconsidered positive (37).
36
2.4.2.8Listeriaspp.
IsolationofListeriamoaocyeogeneswas conductedas perprotocols outlinedin HPB MFHPB-30.Thismethodisbased on the USFDAme thod withmodificationsbased on researchbyWarburton eral (18).
Approxima telyI1.0 g samplesofcaviar were stomac hedinthe Stomache rLabBlender (CanlabDivision,BaxterCo rpo ratio n. MountPearl,NF)for I minutewith 99ml of Listeria enric hment broth (LEB). LE B cultureswereincubatedinthe stomacherbag at 30"Cfor 48 hours.At 24and 48 hours,the LEBculturewasmixed and streaked onto Oxfordagar (OXA) and PALCAM(PAL). Plates were incubatedat35"Cfor24-48hO UTS. The inoc ulat ion ofModifiedFraserbrothstepwasnotundertaken(I8).
Plates wereexaminedfortypicalLmonocytogenesgrowth characteristics . There were no colon iesindicativeofL monocytogenesontheplates,thusL.m01l0cytogc'!lleSwere conside redabsent.Therefore,itwasnotnecessary to cond uctide ntification,confirmatio n andserologica lanalysesfor1_monocytogenes.
37
2.5 Isolation of Predominant Organism
The predominant organisms which accounted for approximately 90% of all organisms wasisolated and purified.The caviare isolatewas grown in Trypticase Soya Broth for 24 hoyurs at 30"C.These culturesserved as the inocula for the growth profiles.
2.6 Growth profiles ot Cavtare Isolate for Various Preservative Methods
Growth profiles(determined by optical densities )of the caviare isolate under various preservativemethods (salt, temperature. sodium benzoate,potassiumsorbate,and a sodium benzoateJpotassiumsorbate mixture)were examined. Series of test tubes containing nine mLoftrypticase soya brothwithvaryi ng concentrations of the preservativeswere prepared.
The tubescontaining the chemical preservatives sodium benzoateand potassiumsorbate werepHadjustedto pH 4.0,5.0, 6.0 and 7.0from an initial pH of7.:!..The pH adjustment was achieved bythe addition of 0.1 N HCI. The pH of the solutions weretestedprior to sterilization. Anadditionaltube was prepared to test the pH of the solutions after sterilization. All solutions maintained the pre-sterilization pH within 0.2units and no additionaladjustmentwasmade.
38
Six tubesforeachseries were inoc ulatedwith l rot of theinocula.Theinoc ula was enumera tedbypreparing serial dilutions up toio-dilution.Thesedilutions were platedby the spread platemeth od on to pre pare d platesofstandard method agar.Theplates were incubatedat 30°Cfor48ho ursandenumerated.
2.6.1 Tempe rature
Seriesof testtubes( 8 testtubes) conta ining9 mloftypticasesoyabroth were prepared.
Oneml ofinoculum was dispensed intoeachtest tube usinga Eppendorfpipetterwith sterile tips.Theinoculaweredispensedintothe tubesimmediately abovethe solutiontaking care not to touchthe solution.Tubesweremixed on avorte x:mixer.Theinitial opticaldensities weretake n usingone tube fromeachseries.The opticaldensit iesweredeterminedbya Shimatzudoublebeam spectrophotometerata wavelengthof630tun.Anuninoc ulated tube (atthe concentratio nbeingtested)wasused to zerothe spectrophotometer.
The remaining six:tubes (one uninoc ulated tube) wereincubated for 24 hoU1'5 at 30°C.
The opticaldensitieswererecordedandtheresultscorrected for theinitia linoc uladensity.
39
2.6.2 Salt
Series of test tubes (8 testtubes ) containing9ml of typticasesoya broth withsalt concentrationsranging from 0to 24% were prepared. Six tubes per saltconcentrationwere prepared;onetube forinitialinocul umreadingand fivereplicate samples.
Onemlof inoculawas dispense dinto eachtest tube using a Eppendorfpipetterwith steriletips.Theinoculawasdispensedinto the tubes immediatelyabove the solutiontaking care notto touch the solution.Thetipwaschanged foreach concentration to ensurethat therewasno carryover of solutionfrom a differentconcentration.Tips werechangedif the solutionwastouched Tubeswere mixed ona vortexmixer.The initial opticaldensities weretakenusing one tube from eachsaltconcentration. Theopticaldensitieswere determinedby a Shimatzu doublebeam spectrophotometerat a wavelengthof 630 nm.An uninoculated tube (at the conce ntration beingtested) was used to zero the spectrophotometer.
The remaining six tubes(oneuninoculatedtube)wereincubatedfor 24 hours at30eC.
The opticaldensitieswere recordedand the resultscorrected for theinitial inoculadensity.
z.o
Growtb Profile(or SodiumBenzoa teSeriesof testtubes(8 testtubes)containing9 mloftrypticasc: soyabrothwithsodium benzoateconcentrations
ero.
250, 500,750and 1000ppmwereprepared at pH'sof4.S.6, and7.The pH adjustmentwas achievedbytheadditionof 0.1NNaOH. The pHofme solutionsweretestedpriortosterilization andallpH' swereconfirmedafter sterilizationby testing the pHof one tube.Allsolutions maintainedthepre-ste rilization within0.2units.No additional pHadjustmentwasnecessary.
Oneml of inocu lumwasdispensed intoeachtesttube usinga Eppendorfpipenerwith sterile tips.The inoculumwas dispensedinto the tubesimmediatelyabove the solution takingcare notto touchthe solution.The tipwaschanged foreach concentrat ion to ensure thattherewasno carryover of solutionfromadifferent co ncentration, Tipswerechanged ifthesolutionwastouched Tubeswere mixedon a vortexmixer. The initialoptical densities weretaken using onetube from eachsodiumbenzoate concentrationperpH level.
The opticaldensitiesweredeterminedbyaShima tzudoublebeamspectrophotometerat a wavelengthof630run.AnuninocuJatedtube (attheconcentration being tested)wasused to zerothe spectrophoto meter.
Theremainingsix tubes(one uninoculatedtube)wereincubated for24hoursat 30°C.
Theopticaldensities were recordedandthe results corrected fortheinitialinocula density.
41
2.6.4 Growt h ProfileforPo ta ssium Sor bate
Series oftest tubes (8 testtubes) containing 9 mloftrypncasesoya brothwithpotass ium sorbateconcentrationsof 0,250, 500, 750and1000 ppmwere prepared atpH's of4.5.6, and7. The pH adjustmentwasachieved by theadditionof 0.1 N NaOH.ThepH of the solutionsweretestedprior to sterilizationand all pH'swere confirmedaftersterilizationby testing the pH of onetube.Allsolutions maintainedthepre-sterilization within0.2units.
No additionalpHadjustment wasnecessary.
Oneml ofino cula was dispensedinto eachtesttubeusing a Eppendorfpipetterwith steriletips.Theinocul a was dispensedintothe rubes im mediatelyabove the solutiontaking ca re not totouchthesolution.The tipwaschangedforeach concentratio nto ensurethat therewasno carryoverof solutionfrom a differentconcentration.Tipswere changed if the solution wastouc hed.Tubes were mixedonavortex mixer.Theinitial optical den sities were taken using one tube fromeac h potassium sorbate concentrationperpH level. The optical densitiesweredetermi nedby aShimatzu doublebeamspectrophotometerat a wave length of 630nrn.Anuninoculatedtube(atthe concentrationbeingtested) was used to zero the spectro photometer.
Theremainingsix tubes(oneuninocul ated tube)were incuba tedfor 24 hours at30°C.
The opticaldensitieswererecordedand the resultscorrectedforthe initialinoc u la density.
42
2.6.5 Growth Profile for Sodium BeDZoateIPotassium Sorbate Combinat ion Prese rvatives
Series oftesttubes (8 testtubes)containing9 mlof trypticese soya broth withsodium benzoate/potassiumsorbate at a ratioof I:I wereprepared.The final concentrationswere 0.250, 500,750and 1000ppm atpH'sof 4,5,6,and 7.The pH adjustmentwasachieved bythe additionofO.1N NaOH.The pH of the solutions were testedprior to sterilizationand all pH'swere confirmed aftersterilization by testing the pH ofone tube. Allsolutions maintainedthe pre-sterilization within 0.2units. No additionalpH adjustment was necessary.
One ml ofinoculawasdispensedinto each testtube using aEppendorf pipenerwith sterile tips.Theinoculawasdispensedinto thetubes immediatelyabovethe solutiontaking carenot totouch thesolution. The tipwaschanged for each concentration toensurethat therewas no carryoverof solution fromadifferentconcentration.Tipswerechanged if the solutionwastouched.Tubes weremixedon avortex mixer.The initialopticaldensities were taken usingone tube from eachsodium benzoate concentrationper pHlevel. The opticaldensitiesweredeterminedbyaShimatzu double beamspectrophotometer at a wavelengthof630nm.Anuninoculatedtube(at the concentrationbeing tested)wasused to zero the spectrophotometer.
43
The remaining sixtubes (one uninoculated tube) wereincubated for 24 hours at30°C.
The optical densitieswere recorded and the resultscorrected for the initialinocula density.
2.7Statistical Analyses
Statisticalexaminatio n of the data wasconducted usingthe computerstatistical package Systatfor windows ?" , Version5. Thestatistica l analyses cond ucted were Analysisof Variance (ANOVA)and pairwisecomparisons wereconductedbyBonferroni Adjustment.
Analysesof variance which isa classical stati stica l techniq ue for analysin gdata which has a quantitativedependent variable and a categoricalindependent variable was conducted on data collecte d for aerobicand anaerobic stan dar d platecounts at normal and abusive sto rage temperaturesusing theSystat program(42).TheANOVAprocedure comparesdifferences in means(42). ANOVA techniquescomputethe variabilityofeachdependentvalue score from the"grand mean"ofscores(42),
A pairwisecompari sonofthedata usingBonferroni ' s Adjustme nt provides information which identifi esthestatistica l difference sbetween preservatives. The ANOVA tellsus there are differenc es in the data and thatit is dueto thepreservativemethods. However,it does not tell us whereor whythedifferences with the data are occurring.TheBonferro ni Adj ustmentisa strong asset in making comparisons amongsimple pairs of means(42).For exam ple, we are evaluatinga number ofpreservativemethods andwe want todetermine
44
which preformssignificantlybetterthan theothers.Therefore,we wishto compare differences among all possible pairs of level means. To achieve this endwecan applythe BonferroniAdjustment or Tukey HSDtest. TheBonferroniAdjustment tendsto be more rigorous than the Tukey HSDTest. Initiallyboth the Bcnferrcni Adjustment and the Tukey HSD were conduc tedandthere were no differencesbetweenthe two results obtained.Thus the Bonferroni Adjustmentisreportedas itisconsideredthe morerigorous of the two methods.
45
CHAPTER J RES ULT S
All samples were storedat 20"Cto approximateroom temperature and37"Cas an abusivetemperature.Thechoiceof the2Q"Ctemperature was based on the assumption that retail outletswouldtrytomaintaintheir temperaturesat ornear 20"C. The abusive temperature of 37"Cwaschosensince cargo containers,retailstorageareas. andthe South Koreanclimate could reach temperaturesnear37"Cunder certainconditions.Refrigerated samplesweresto red at 4"C(nonnalconditions)andlOGe (abusive conditions).
Samples wereexamined for chemicaland microbiological qualityovera four month timeperiod. Thesampleswere analysedat0,14,28.56.and112 days.Rawmaterialused inthe experimentalrunswerealso examined. Three sampleswereanalysed in duplicatefor allmicrobiologicaland chemical parameters.
46
3.1 Cbe mica lAa aly5eS
The chemicalparametersexaminedwere:
pH.
2. Sale
3. Wateractivity.
4. Ash.
5. Moisture.
6. Fae
7. Protein.
The results forthechem ical analysesarelocatedinAppendixA (TablesAI-AI4 ).The chemical parametersshowed littleor novariationove rtime(Table3.1).
Table3.1: Proximate AnalysesResults
Parameter Range Mean±Standard Deviation
pH ';.97-6.2'; 5.902<O.I:H
Salt(%) ';.95 · 7.14 5.88±0.35
Moisture(%) 74.56- n.52 74.93±1.00
WaterActivity 0.920-0.939 0.934±0.003
Ash(%) 5.20-6.95 5.92±0.49
Fat(%) 1.28-4.09 2.15±0.4 1
Protein (% ) 11.21· 15.6 1 13.88±0.81
47
3.2Mi~robiologicalAna lyses
The microbialanalysesconductedwere:
I) Aerobi c platecounts. 2) Anaerobic platecounts.
3) Anaerobicsporulative counts.
4) Tota l colifo rm.
5) Fecal colifo rm.
6) Coa gulase positi ve Staphylococcusuureus.
7) Salmo ne llaspecies. 8) Bac illuscereus.
9) Liste riamonocyt ogenes.
The resultsforallmicrob ial analysesconducted are presented in Appendix 8(TablesBI -816). Bscdius cereus,LWt!ri<lnlU"Ul.ytug"mt!~.StupllylUf.:Vf..l:w uureus,Sulmundlu
species.andEscher ichiacoliwerenot detected in anysamples.Thisindicatesthatthe lwnpfishroe caviarwasfreeof a majority ofthe majorfood pathogens.The onlyfood pathogens of worrywouldbe anaerobic fonnssuchasCloarida spp..Total coliforms when detectedwereusually below500 cfu pergramand notconside red a problem.
48
3.2.1 Prese rvativesandSto rageCond itio ns
Bacterial growth in caviare samples exhibitedthe same pattern of growth regardlessof the preservative regime(F igures3.1 - 3.8).Bacterialloads increased fromtheinitialload (day0)to reach maximum bacterial loadsbetwee n 14-28 days., then fellback:to levels near the initial loads.The final baeteri al loa ds (day 112) were more varaiblethan anyotherday patte rn onthebacterialgrowth curves.Some preservative regimeshadfinalbacterialloads that were slightlyhigherthanthe initia lbacterialtoads.others hadessentia llythe same final toadas theinitial load,andothers werelowerthan the initialloads. The re wasno discernablepatte rn tothefinalloads basedonstoragetempe rature ofpre servativemethod.
Temperatureprese rva tio n techniques(pasteurization and refrigeration)generally achievedthehighest bacterialloads earlier inthegrowth curvethandid samples that were chemicallypreserved.Samples stored at200cgeneTa!lyachievedthe highest baeterialloads earlierthansamples storedat37'C.
Graphic presentatio n ofresults for aero bicandanaerobic platescounts (logtransfonned)are presentedin Figures 3.1- 3.8.These Figures showthat pasteurizationat 55°Cand sodium benzoa te(SOmglkg) arenot effectivepreservati onmethodswithrespect to eitheranaerobe oraerobicplates counts.
49
Figure Legend for Figure 3.1.
Figure Legend Identification Explanation
Sodium BenzoateIPotassium Sorbare 3:1 Sodium Benzoate(300 ppm)and Potassium Sorbate (100 ppm) mixture.
Sodium Benzoate/PotassiumSorbate 1:1 Sodium Benzoate (200 ppm) and Potassium Sorbate(200 ppm)mixture.
Sodium BenzoateIPotassium Sorbete U Sodium Benzoate (100 ppm) and Potassium Sorbate (300 ppm) mixture.
Sodium Benzoate Sodium Benzoate (80 ppm) - Present
commercial product.
50
~
l'.u;
I I I -
Q~;.
..2~
~ ~ ~
~]
'"
.. ';:i i i
§ § §:e
EcI .. ~.
i Ii Ii Ii Ii " 'li j i ~ I~'" I - g
~ ~
i=~~e
..2J: ~ §
2t.Jg
~ ~ ] ]
~
8
+ + + + -
Q~ ~E~
;:; e
ii:
...
Figure legendfor Figure 3.2.
Figure Legend Identification Pasteurized 55°C
Pasteurized 70°C
Refrigerated
Explanation
Sampleswere pasteurized at55°C for 135 minutes.
Sam ples were pasteurized at 70°Cfor 45 minutes.
Samples were stored under refrigerated condition sat 4°Cwith neitherpasteurization orchemicalpreservatives.
52
s
_ C
N
g
0 0
Figurelegendfor Figure3.3.
figure Legend Identification Explanation
Sodium BenzoateIPotassiumSorbate3:1 SodiumBenzoate(300 ppm)and Potassium Sorbate(100ppm) mixture.
Sodium BenzoateIPotassiumSorbate1:1 SodiumBenzoate(200ppm)and Potassium Sorbate(200ppm) mixture.
SodiumBenzoate'PotassiumSorbate1:3 Sodium Benzoate(100ppm) and Potassium Sorbate(300 ppm)mixture.
SodiumBenzoate Sodium Benzoate (80 ppm ) - Present
commercial product.
54
I 112Days 56Days
14Days
-+-SodiumBenzcnte/PotessiumSorbate3:I(400 ppm) ___SodiumBenzoate/PotassiumSorbate1:1(400ppm) -.-SodiumBenzonteIPo18ssiumSorbarel.J(400ppm) -it-SodiumBenzoate(80 ppm)
I
28Days Time (Days)
Figure3.3:Aerobic<:ounllror ChemicalPreservationmethodsror AbusiveStorage Conditions.
o
OOays
~ 6 o
8.
5~
'" 4
·2 :::J
j i!!'
g
8 .3
Figure legendforFigure 3.4.
Figure LegendIdentjficatio n Pasteurized 55°C
Pasteurized70°C
Refrigerated
Explanation
Sampleswerepasteurizedat 55°Cfor 135 minutes.
Sampleswere pasteurizedat 70°Cfor 45 minutes.
Samples were stored under refrigerated conditionsat 4°Cwith neitherpasteurization or chemical preservatives.
56
Figurelegendfor Figure3.5.
FigureI egend Identificatio n Explanation
SodiumBenzoate/PotassiumSorbate 3:I Sodium Benzoate(300 ppm)andPotassium Sorbate (100 ppm) mixture.
Sodium BenzoateIPotassiumSorbateI:1 SodiumBenzoate (200ppm) and Potassium Sorbate (200 ppm)mixture.
SodiumBenzoateIPotassium Sorbatel:3 Sodium Benzoate(100 ppm)and Potassium Sorbate(300 ppm)mixture.
Sodium Benzoate Sodium Benzoate (80 ppm)- Present
commercialproduct
58
g
~ R.
.~ "
I g
8 .3
o
o Days
FigureJ.S:
-.-Sodium BenzoateIPotassiumSorbate3:1(400ppm) __SodiumBenzoateIPotassium Sorbate1:1(400 ppm) -.-SodiumBenzoate/PotassiumSorbate l:3(400ppm)
-M-SodiumBenzoate(80ppm)
! ....- 1-
14 Days 28 Days S6Days 112 Days
Time (Days)
AnaerobicCount,IerChemln lPreservancnMethodsrorNormalStorage Conditions.
Figure legend forFigure 3.6.
FigureLegend Identificatio n Pasteurized55"e
Refrigerated
Explanation
Sampleswerepasteurized at 55°efor 135 minutes.
Sam ples were pasteurized at 700
e
for 45minutes.
Samples were stored under refrigerated conditions at 4°Cwithneither pasteurization or chemical preservatives.
60
