Sterility 63601 Tetra Pak Document

 An introduction to commercial sterility

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Index  3 4 4 5 7 8 9

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Introduction Histor y Single-cellriendsandenemies Thelogarithmicorder Approachingabsolutesterility Aweaklinkbreaksthechain Meetingmarketrequirements

10 11 12 13 14 15

Howmanysamples? Increasingsterilizationeciency Amixtureoremedies UHTtreatment Microltration Summar y

Introduction Thepurposeothisbookletistoprovideabasicunderstanding othe microbiologicalaspects oaseptic technologywiththeocusontheprocessingpart.We donotaimtocovereverythingindetail,butinsteadtry togiveyouagoodoverviewotheimportanceocommercialsterilityandhowtoachieveit.Iyouneedmore detailedinormation,donothesitatetocontactusat TetraPak.Wearehappytoassistyouwithourcompetenceandexperience.

Allqualityisbasedoncompetence.

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History The destruction o microorganisms EvenbeoreLouisPasteurprovedoveracenturyago thatmicroorganismscausedermentationanddisease,NicolasAppert,aParisianconectioner,irst succeededinpreservingcertainoodsinglassbottles thathadbeenkeptinboilingwaterorvaryinglengths otime.Thishappenedintherstdecadeothe1800’s

andby1839,tin-coatedsteelcontainerswerewidelyin use.Thusbeganthebirthohighheattreatmentand canningasameansopreservingood. Today,acombinationocontinuousheat-treatment andasepticpackagingproduceshighqualityliquid oodinacost-eectiveway.

Single-cell riends and enemies SporesposethemajorchallengetoUHT(ultrahigh temperature)processinganddeterminethesterilizationparameters. Microorganismscarryoutinnumerableunctions thatareessentialtolieonearth.However,asregards oodproducts,theyarepublicenemynumberone. Manybacteria,someowhicharepathogenictoman, areresponsibleortherapidspoilageoood.Applying asuitabletemperature(e.g.72°Cor15s)degrades

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proteins,essentialcomponentsolivingcells,andsoit isquiteeasytokillvegetativebacteria. Themajorproblemisbacterialspores.Theseare diculttokillbecausetheyhavethickprotectivewalls whichincreasetheirresistancetoheat,drying,reezing,chemicalagentsandradiation.Thisiswhyspores posethebiggestchallengetoUHTprocessinganddeterminetheparametersusedorsterilization.

The logarithmic order  A never-endi ng story Asepticprocessinginvolvesoneormoreseparatesterilization stepsaimed at killingall microorganisms. Whenapplyinga sterilizationprocedure,e.g.using heat,notallbacterialsporesarekilledatthesametime. Acertainnumberwillbekilledwithinagivenunito timeasshowninthetablehere.Theprinciplebehind thekillingprocessisindependentothesterilization temperature. Whenthenumberosurvivingsporesisplotted againsttheirlogarithmicreduction,weobtainastraight lineasshowninFigure1.Thisstraightlineiscommonly knownasthelogarithmicorderodeath,andtheslope othelineindicatestherateodestruction.

1000000

NUMBER OF SPORES

100000 1000 100 10

D

1 0.1

1

2

3

4

5

1.01 0.001 TIME

0.0001

Fig. 1. A typical graph o the logarithmic order o death or bacterial spores during sterilization. The steeper the slope, the higher the rate o killing efciency. The initial microbial load is here assumed to be 100 000 bacterial  spores o equal resistance. D is the decimal reduction time. UNIT OF TIME

SURVIVORS

0 1 2 3 4 5 6 7 8

100 000 10 000 1 000 100 10 1 0.1 0.01 0.001

SPORES KILLED PER UNIT TIME

0 90 000 9 000 900 90 9 0.9 0.09 0.009

TOTAL ACCUMULATED

0 90 000 99 000 99 900 99 990 99 999 99 999.9 99 999.99 99 999.999

LOGARITHMIC REDUCTION

0 1 2 3 4 5 6 7 8

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 Approaching absolute sterility Asalogarithmicunctionnevercanreachzerobutonly approachit,absolutesterilitynevercanbereachedor guaranteed.Theeciencyoasterilizationprocessis expressedbythenumberodecimalreductionsinthe countoresistantspores.TheD-value(decimalreductiontime)isnormallyusedtoindicatethekillingrate, i.e.thetimerequiredatacertaintemperaturetokill 90%othespores,whichisthesameasreducingtheir numberbyonelogarithmunit.Measuredunderthe sameconditions,theD-valuewillbeaectedbythe speciesobacteria.

The result o a heat sterilization process depends on: •theinitialnumberomicroorganisms; •thedecimalreductiontime(heat-resistance) omicroorganisms; •thetimeandtemperatureoheatexposure. AnotherimportantparameteristheZ-value, whichisthechangeintemperatureneeded toaltertheD-valuebyaactoroone logarithmunit. D-VALUE

Absolutesterility canneverbereached orguaranteed.

1000 100 10 Z

1 0.1

TEMP °C

120

130

140

150

160

Fig. 2. The Z-value is the increase in temperature needed to achieve  the same lethal action or the same eect in one-tenth o the time.

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 A weak link breaks the chain Asepticprocessingandpackagingisrathercomplex becausea minimum othree dierentsterilization stepsare required toachieveaseptic llingo the liquidoodproduct.Sterilizationiscarriedout: • • •

ontheequipmentpriortoproduction; ontheliquidoodproduct; onthepackagingmaterialsurace.

Theperormanceleveloanasepticplantmustberegardedastheperormancelevelotheentireprocess ratherthanthatoasinglecomponentotheproductionline.Eachlinkintheprocessingandpackaging chainmustbeequallystrong.

Eachlinkintheprocessingandpackaging chainmustbeequallystrong.

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Raw  material

Preprocessing

Packaging material

UHT treatment

Sterilization

 Aseptic packaging

Meeting market requirements Theprocessingresultisnotdeterminedbytheprocessingactorsalonebutalsobythequalityorawmaterial,pre-processingprocedures,environmentaland operativeparametersaswellasmaintenance.Absolute sterility–denedasthetotalabsenceomicroorganisms–isnotpossibleorthereasonmentionedearlier andthuscannotbeguaranteed. Commercial sterility AccordingtotheWHO/FAO,commercialsterility olow-acidoodisdenedasollows:

“Commercial sterility means the absence o microorganisms capable o growing in the ood at normal non-rerigerated conditions at which the ood is likely to be held during manuacture, distribution and storage.” (Re: Codex Alimentarius Commission (WHO/ FAO) CAC/RCP 0-)

TheEUdenestheheattreatmentnecessaryto achievingcommercialsterility.

“UHT treatment is achieved by a treatment: (i) involving a continuous ow o heat at a high temperature or a short time (not less than °C in combination with a suitable holding time) such that there are no viable microorganisms or spores capable o growing in the treated product when kept in an aseptic container at ambient temperature, and (ii) sufcient to ensure that the products remain microbiologically stable ater incubating or  days at 0°C in closed containers or or seven days at °C in closed containers or ater any method demonstrating that the appropriate heat treatment has been applied.” (Re: Commission Regulation (EC) No /00 (amending Regulation (EC) No /00)).

Speciying quality levels Thepracticalconsequencesoabsolutesterilitybeing unobtainable means establishing microbiological specicationsortheend-product.Thisisnecessaryin ordertodevelopaviablequalitycontrolprogramme. Suchspecicationsoracceptancequalitylevels(AQL) ormanintegralpartoacompany’squalitypolicy. Amicrobiological AQLor long-lieproducts shouldbeexpressedasamaximalacceptabledeect rate.Acleardistinctionhastobemadebetweenthe maximalacceptabledeectiveratesandthegoalo production.Regardlessoestablishedmaximalacceptabledeectrates,thegoaloproductionmustalways beperection. Sampling procedure Amaximalacceptabledeectrateoaround1per1000 to10000samplesisgenerallyapproved.Inorderto detectsuchdeectrates,samplingstatisticsmustbe introduced.Thisincludesdeningtheleveloprobability(condencelevel)tobeusedwhencheckingor deectrates.Usually,a90–95%condencelevelo achievingacorrectresultisused.

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How many samples? More samples will improve the detection level Todeterminehowmanysamplesweneedtotaketo detectadeectiveproduct,let’slookatatypicalexample.Howmanysamplesarerequiredtodetect adeectrateo1per1000with90%probability? Thisproblemissolvedwiththehelpotherandom samplingcurveor90%probabilityshowninFigure3. Iweollowthe1%gureuptowherethecurveintersectsthey-axiswegettheresult240.For0.1%(1per 1000)theanswertothequestionis2400.Thestatistical resultsareaectedonlybythesamplingsizeandnot bythetotalnumberounitsproduced. Let’stakeanotherexampleusingthesame90% probabilitycurve.Iwetake50samplesromabatch andnoneothemaredeective,whatisthedeect rate? Inthiscasewetakeahorizontallinerom50samplesonthey-axis,andwhereitintersectsthe90% curveisthedeectrateatthisleveloprobability:less than5%,orabout5samplesper100units.Moresampleswillimprovethedetectionlevel. Todetectadeectrateo1per1000with95% probability,wewouldneedtotake3000samples.

0

NUMBER OF SAMPLES

300

200

100

0

DEFECT RATE %

1

1.5

2

3

4

5

6

7 8 910

Fig. 3. The relation between deective product samples in percent and the total number o sampl es to be analysed  or bacteria needed to fnd one or more deects i n samples at 90 and 95% levels o probability.

 Analy tical me thods Therearemanymethodsavailableordetectingbacteriaandestimatingtheirnumber.Themostcommon onesaregiveninthetablebelow. Analytical method Bacteriologicalplating BacterialATP Sensory pH

Accuracy ••• ••• •• •

Cost •• ••• • •

Highlysensitivemethodsdetectthemostbacteriabut arealsothemostexpensive.Inbacteriologicalplating, asampleoliquidoodisspreadoutonnutrientagar platesandanycoloniesthatariseromindividualbacteriaaterincubationarecountedtoestimatetheconcentrationobacteriaintheproduct.Severalmethods areavailablethatdisruptthebacterialwallsothata compoundcalledadenosinetriphosphate(ATP)present invegetativecellscanleakoutandbemeasuredto estimatethenumberoviablebacteria.SensoryandpH methodsarethecheapestbuthavepooraccuracy. Whichmethodischosendependsonabalance betweenthemaximalacceptabledeectrate,analytical costandthecompetenceocompanypersonnel. AccordingtotheEuropeanCouncilDirective,random samplesoUHTmilkmustbecheckedusingthebacteriologicalplatecounttechnique,althoughthenumber osamplesisnotstated.

Thestatisticalresultsareaected onlybythesamplingsizeandnotby thetotalnumberounitsproduced.

Increasing sterilization efciency Theeciencyoasterilizationprocesscanbeexpressed asthenumberodecimalreductionsinmicrobial populationachievedbytheprocess. This is mainly determined by two actors: 1. Thetemperatureandthelengthotime i t i s a ppl ie d. 2. Theheat-resistanceobacterialspores present,D-value. Someotheractors,e.g.thecomposition,viscosity,uniormityandpHotheliquidoodproduct,willalso aectthesterilizationeciency.FortypicalUHTprocessingequipmentitcanbeassumedthatanaverage o9–10decimalreductionscanbeachievedwithbacterialsporesgrowingatambienttemperatures.

Initial spore load /ml 10000=10 4 100=102

Spore load/l 107 105

UHT decimal reduction 9D 9D

Spores/l in product 0.01 0.0001

More spores, more problems Inadditiontotheactorsmentionedabove,theeciencyotheUHTprocessisinfuencedbythespore contentotheliquidood,i.e.thetotalsporeload,ed intothenalheat-treatmentsectionotheUHTplant. Whatistheeectodierentinitialsporeloadson end-productquality,assumingidenticalprocessing conditions?Itheproductislledinto1-litrepackages, thedeectratecanbedeterminedromtheinormationgiveninthetable.

Defect rate 1:100 1:10 000

NUMBER OF SPORES

High temperature Low temperature

105 104 103 102 101 10 0 10 -1 10-2

Figure4illustratestheeectoincreasedsterilization temperatureoninitialsporeload,andtheeectodierentsporeloadsonthesametemperature.

Sterilizationeciencycanbeexpressed asthenumberodecimalreductionsin microbialpopulation.

10 -3 10 -4 10 -5

TIME

Fig. 4. Increasing the temperature or a particular initial  spore load achieves a quicker killing rate. Increasing the  initial spore load or a particular temperature leads to a longer time required to achieve the same spore reduction as or a lower spore load.



 A mixture o remedies Theimportanceoinitialsporeloadonsterilization eciency,andhenceend-productquality,isobvious. Butwhatcanbedonetoimprovethesituation?The mostimportantmethodsoachievingthisaregiven belowandsummarizedinthetable. Improved hygiene Ahighconcentrationosporesmayalreadybepresent inmilkatthearm,orbeintroducedintorawmaterials asaresultopoorpreprocessingroutines.Theanswer hereistoimprovethelevelohygienebothatthearm andtheprocessingplant.However,itisdiculttocontrolwhathappensonaarmanditisnotalwayspossibletochangemilksuppliers. Spore reduction Agoodwayoimprovingtheend-productistoreduce thesporecontentorawmaterialsbeoretheliquid oodisheat-treated.Processessuchasbactougation andmicroltrationarespeciallydesignedtogreatly reducethesporecontentomilk. Eective heat treatment Anincrease intemperatureand/orprolongingthe productholdingtimeduringheattreatmentwillresult inamoreeectivesterilizationprocess.Asaconsequence,thenumberodecimalreductionswillincrease andthedeectlevelwilldecrease.



Spore reduction remedies Process Milking Preprocessing Sporereduction UHTtreatment

Remedial action to reduce spore count Improve arm hygiene Change suppliers Improve plant hygiene Bactougation,microltration or other measure Increasetemperature Prolong holding time

SporereductionandimprovedUHT treatmentarethemosteectivemeans oincreasingsterilizingeciency.

Ef fectiveness Helpul but hard to control Helpul Eective Very eective

UHT treatment Two principles TherearetwodierentUHTmethodsusedinthe liquidoodindustry– directand indirect heating. Whichonetousedependsonanumberoactor ssuch asdesiredendproductqualitylevelandproduction economy. Direct heating Whenusingdirectheating,theproductisbroughtinto directcontactwithhotsteamunderstrictlycontrolled conditions.Thesterilizationtemperature(140°Cor4 sec)israpidlyreachedandaterholding,thetemperatureisloweredbyfashcoolinginavacuumvessel.The rapidheatingandcoolinggivesminimalheat-loadon theproductwithahigh-qualityproductastheresult. Indirect heating Inthismethodapartitionisplacedbetweentheproductandtheheatingorcoolingmedium.Theprinciple isthatahotmediumisfowingononesideothepartitionandtheproductontheother.Thepartitionis heatedupandtranserstheheatovertotheproduct lowwithoutanydirectcontactbetweenthehot mediumandtheproduct.Thesameprincipleapplies tocooling.

Thisprocedureisdoneinwhatisreerredtoasaheat exchanger.Whichonetochoosedependsonseveral actors,e.g.productfowrate,physicalpropertieso theliquid,requiredrunningtime,cleaningrequirementsetc. Theollowingthreetypesoheatexchangersarethe mostwidelyused: • Plateheatexchangers(PHE)aremainlyused orsmoothlowviscousliquidproducts. • Tubularheatexchangers(THE)canalsohandle productswithparticlesuptoacertainsizeand oerlongerrunningtimes.Themaximumsize oparticlesdependsonthediameterothetube. • Scraped-suraceheatexchangers(SSHE)are speciallydesignedorheatingandcoolingo viscous,stickyandlumpyproducts,withor withoutparticles. TheeciencyandfexibilityoUHTprocessingarecontinuouslybeingimproved.OnetypeoUHTmodule incorporatesboth directand indirect heating with tubularheatexchangerstoprovidedierentcombinationsoheattreatmentinonesystem.



Microfltration AcombinationomembraneltrationandUHTtreatmentisproventobethemosteectivemethodo increasingsterilizingeciency. Byusingamicroltrationmembrane,99.5-99.95% obacteriaandsporesareeectivelyremovedinacostecientway.Thisisachievedbylettingtheliquidsubstancepassoveramembraneathighspeedunder pressureinaclosedsystem. Themembranehasveryspecicpropertiesthat onlyallowthebacteriaandsporesoa“maximumsize” topassthroughthemembrane.Sincethesizeand weightotheunwantedbacteriaandsporesareknown, thelterusedisconstructedwithappropriatepore

sizestoremovethesepartsromtheliquidandletthe smallerprotein,lactose,mineralandwatermolecules passontonalUHTtreatment. Themainbenetsothisprocedurearethatthe productwillbemorepuriedbeoreUHTtreatment andthenalproductwillnotcontainanydeadspores. Thisgivesanend-productwithhigherquality,better tasteandlongershel-lie.

Microltrationminimizesthebacterialandspore contentomilkinacost-eectiveway.



Summary • SporesposethebiggestchallengetoUHTprocessing anddeterminethesterilizationparameters. • Absolutesterilitycanneverbereachedorguaranteed. • Eachlinkintheprocessingandpackagingchainmust beequallystrong. • Acceptancequalitylevels(AQL)ormanintegralpart oacompany’squalitypolicy. • Statisticalresultsareaectedonlybythesamplingsize andnotbythetotalnumberounitsproduced. • Moresampleswillimprovethedetectionlevel. • Sterilizationeciencycanbeexpressedasthenumber odecimalreductionsinmicrobialpopulation. • SporereductionandimprovedUHTtreatmentarethe mosteectivemeansoincreasingsterilizingeciency.



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