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An introduction to commercial sterility

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3 Introduction

4 History

4 Single-cellfriendsandenemies

5 Thelogarithmicorder

7 Approachingabsolutesterility

8 Aweaklinkbreaksthechain

9 Meetingmarketrequirements

10 Howmanysamples?

11 Increasingsterilizationefficiency

12 Amixtureofremedies

13 UHTtreatment

14 Microfiltration

15 Summary

Index

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Thepurposeofthisbookletistoprovideabasicunder-

standing of the microbiological aspects of aseptic

technologywiththefocusontheprocessingpart.We

donotaimtocovereverythingindetail,butinsteadtry

togiveyouagoodoverviewoftheimportanceofcom-

mercialsterilityandhowtoachieveit.Ifyouneedmore

detailedinformation,donothesitatetocontactusat

TetraPak.Wearehappytoassistyouwithourcompe-

tenceandexperience.

Introduction

Allqualityisbasedoncompetence.

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History

The destruction of microorganismsEvenbeforeLouisPasteurprovedoveracenturyago

thatmicroorganismscaused fermentationanddis-

ease,NicolasAppert,aParisianconfectioner,first

succeededinpreservingcertainfoodsinglassbottles

thathadbeenkeptinboilingwaterforvaryinglengths

oftime.Thishappenedinthefirstdecadeofthe1800’s

Single-cell friends and enemies

Sporespose themajorchallengetoUHT (ultrahigh

temperature)processinganddeterminethesteriliza-

tionparameters.

Microorganismscarryout innumerablefunctions

thatareessentialtolifeonearth.However,asregards

foodproducts, theyarepublicenemynumberone.

Manybacteria,someofwhicharepathogenictoman,

areresponsiblefortherapidspoilageoffood.Applying

asuitabletemperature(e.g.72°Cfor15s)degrades

proteins,essentialcomponentsoflivingcells,andsoit

isquiteeasytokillvegetativebacteria.

Themajorproblemisbacterialspores.Theseare

difficulttokillbecausetheyhavethickprotectivewalls

whichincreasetheirresistancetoheat,drying,freez-

ing,chemicalagentsandradiation.Thisiswhyspores

posethebiggestchallengetoUHTprocessingandde-

terminetheparametersusedforsterilization.

andby1839,tin-coatedsteelcontainerswerewidelyin

use.Thusbeganthebirthofhighheattreatmentand

canningasameansofpreservingfood.

Today,acombinationofcontinuousheat-treatment

and aseptic packaging produces high quality liquid

foodinacost-effectiveway.

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The logarithmic order

A never-ending storyAsepticprocessinginvolvesoneormoreseparatester-

ilization steps aimed at killing all microorganisms.

When applying a sterilization procedure, e.g. using

heat,notallbacterialsporesarekilledatthesametime.

Acertainnumberwillbekilledwithinagivenunitof

timeasshowninthetablehere.Theprinciplebehind

thekillingprocessis independentofthesterilization

temperature.

When thenumberof surviving spores isplotted

againsttheirlogarithmicreduction,weobtainastraight

lineasshowninFigure1.Thisstraightlineiscommonly

knownasthelogarithmicorderofdeath,andtheslope

ofthelineindicatestherateofdestruction.

Fig. 1. A typical graph of the logarithmic order of death for bacterial spores during sterilization. The steeper the slope, the higher the rate of killing effi-ciency. The initial microbial load is here assumed to be 100 000 bacterial spores of equal resistance. D is the decimal reduction time.

UNIT SPORES KILLED TOTAL LOGARITHMICOF TIME SURVIVORS PER UNIT TIME ACCUMULATED REDUCTION0 100000 0 0 01 10000 90000 90000 12 1000 9000 99000 23 100 900 99900 34 10 90 99990 45 1 9 99999 56 0.1 0.9 99999.9 67 0.01 0.09 99999.99 78 0.001 0.009 99999.999 8

1000000

100000

1000

100

10

1

0.1

1.01

0.001

0.0001

NUMBER OF SPORES

TIME

1 2 3 4 5

D

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Approaching absolute sterility

Asalogarithmicfunctionnevercanreachzerobutonly

approachit,absolutesterilitynevercanbereachedor

guaranteed.Theefficiencyofasterilizationprocessis

expressedbythenumberofdecimalreductionsinthe

countofresistantspores.TheD-value(decimalreduc-

tiontime)isnormallyusedtoindicatethekillingrate,

i.e.thetimerequiredatacertaintemperaturetokill

90%ofthespores,whichisthesameasreducingtheir

numberbyone logarithmunit.Measuredunder the

sameconditions,theD-valuewillbeaffectedbythe

speciesofbacteria.

The result of a heat sterilization process depends on: •theinitialnumberofmicroorganisms;

•thedecimalreductiontime(heat-resistance)

ofmicroorganisms;

•thetimeandtemperatureofheatexposure.

AnotherimportantparameteristheZ-value,

whichisthechangeintemperatureneeded

toaltertheD-valuebyafactorofone

logarithmunit.

Absolutesterilitycanneverbereachedorguaranteed.

Fig. 2. The Z-value is the increase in temperature needed to achieve the same lethal action or the same effect in one-tenth of the time.

1000

100

10

1

0.1

D-VALUE

TEMP °C

120 130 140 150 160

Z

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A weak link breaks the chain

Asepticprocessingandpackagingisrathercomplex

because a minimum of three different sterilization

steps are required to achieve aseptic filling of the

liquidfoodproduct.Sterilizationiscarriedout:

• ontheequipmentpriortoproduction;

• ontheliquidfoodproduct;

• onthepackagingmaterialsurface.

Theperformancelevelofanasepticplantmustbere-

gardedastheperformanceleveloftheentireprocess

ratherthanthatofasinglecomponentoftheproduc-

tion line.Each link intheprocessingandpackaging

chainmustbeequallystrong.

Eachlinkintheprocessingandpackagingchainmustbeequallystrong.

Raw material

Pre- processing

UHTtreatment

Asepticpackaging

Sterilization

Packagingmaterial

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Meeting market requirements

Theprocessingresultisnotdeterminedbytheprocess-

ingfactorsalonebutalsobythequalityofrawmate-

rial, pre-processing procedures, environmental and

operativeparametersaswellasmaintenance.Absolute

sterility–definedasthetotalabsenceofmicroorgan-

isms–isnotpossibleforthereasonmentionedearlier

andthuscannotbeguaranteed.

Commercial sterilityAccordingtotheWHO/FAO,commercialsterility

oflow-acidfoodisdefinedasfollows:

“Commercial sterility means the absence of microorganisms capable of growing in the food at normal non-refrigerated conditions at which the food is likely to be held during manufacture, distribution and storage.” (Ref: Codex Alimentarius Commission (WHO/FAO) CAC/RCP �0-����)

TheEUdefinestheheattreatmentnecessaryto

achievingcommercialsterility.

“UHT treatment is achieved by a treatment: (i) involving a continuous flow of heat at a high temperature for a short time (not less than ���°C in combination with a suitable holding time) such that there are no viable microorganisms or spores capable of grow-ing in the treated product when kept in an aseptic container at ambient temperature, and (ii) sufficient to ensure that the products remain microbiologically stable after in- cubating for �� days at �0°C in closed con-tainers or for seven days at ��°C in closed containers or after any method demonstrat-ing that the appropriate heat treatment has been applied.” (Ref: Commission Regulation (EC) No ����/�00� (amending Regulation (EC) No ���/�00�)).

Specifying quality levelsThepracticalconsequencesofabsolutesterilitybeing

unobtainable means establishing microbiological

specificationsfortheend-product.Thisisnecessaryin

ordertodevelopaviablequalitycontrolprogramme.

Suchspecificationsoracceptancequalitylevels(AQL)

formanintegralpartofacompany’squalitypolicy.

A microbiological AQL for long-life products

shouldbeexpressedasamaximalacceptabledefect

rate.Acleardistinctionhastobemadebetweenthe

maximalacceptabledefective ratesandthegoalof

production.Regardlessofestablishedmaximalaccept-

abledefectrates,thegoalofproductionmustalways

beperfection.

Sampling procedureAmaximalacceptabledefectrateofaround1per1000

to10000samplesisgenerallyapproved.Inorderto

detectsuchdefectrates,samplingstatisticsmustbe

introduced.Thisincludesdefiningthelevelofproba-

bility(confidencelevel)tobeusedwhencheckingfor

defectrates.Usually,a90–95%confidencelevelof

achievingacorrectresultisused.

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How many samples?

More samples will improve the detection levelTodeterminehowmanysamplesweneedtotaketo

detectadefectiveproduct,let’slookatatypicalex-

ample. How many samples are required to detect

adefectrateof1per1000with90%probability?

Thisproblemissolvedwiththehelpoftherandom

samplingcurvefor90%probabilityshowninFigure3.

Ifwefollowthe1%figureuptowherethecurveinter-

sectsthey-axiswegettheresult240.For0.1%(1per

1000)theanswertothequestionis2400.Thestatistical

resultsareaffectedonlybythesamplingsizeandnot

bythetotalnumberofunitsproduced.

Let’s takeanotherexampleusingthesame90%

probabilitycurve.Ifwetake50samplesfromabatch

andnoneof themaredefective,what is thedefect

rate?

Inthiscasewetakeahorizontallinefrom50sam-

ples on the y-axis, and where it intersects the 90%

curveisthedefectrateatthislevelofprobability:less

than5%,orabout5samplesper100units.Moresam-

pleswillimprovethedetectionlevel.

Todetectadefect rateof1per1000with95%

probability,wewouldneedtotake3000samples.

Analytical methodsTherearemanymethodsavailablefordetectingbac-

teriaandestimatingtheirnumber.Themostcommon

onesaregiveninthetablebelow.

Analytical method Accuracy Cost

Bacteriologicalplating ••• ••

BacterialATP ••• •••

Sensory •• •

pH • •

Fig. 3. The relation between defective product samples in percent and the total number of samples to be analysed for bacteria needed to find one or more defects in samples at 90 and 95% levels of probability.

Highlysensitivemethodsdetectthemostbacteriabut

arealsothemostexpensive.Inbacteriologicalplating,

asampleofliquidfoodisspreadoutonnutrientagar

platesandanycoloniesthatarisefromindividualbac-

teriaafterincubationarecountedtoestimatethecon-

centrationofbacteriaintheproduct.Severalmethods

areavailablethatdisruptthebacterialwallsothata

compoundcalledadenosinetriphosphate(ATP)present

invegetativecellscan leakoutandbemeasuredto

estimatethenumberofviablebacteria.SensoryandpH

methodsarethecheapestbuthavepooraccuracy.

Whichmethod ischosendependsonabalance

betweenthemaximalacceptabledefectrate,analytical

cost and the competence of company personnel.

AccordingtotheEuropeanCouncilDirective,random

samplesofUHTmilkmustbecheckedusingthebacte-

riologicalplatecounttechnique,althoughthenumber

ofsamplesisnotstated.

Thestatisticalresultsareaffectedonlybythesamplingsizeandnotbythetotalnumberofunitsproduced.

300

200

100

0

NUMBER OF SAMPLES

DEFECT RATE %

1 1.5 2 3 4 5 6 7 8 910

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Increasing sterilization efficiency

Theefficiencyofasterilizationprocesscanbeexpressed

as the number of decimal reductions in microbial

populationachievedbytheprocess.

This is mainly determined by two factors:1. Thetemperatureandthelengthoftime

itisapplied.

2. Theheat-resistanceofbacterialspores

present,D-value.

Someotherfactors,e.g.thecomposition,viscosity,uni-

formityandpHof the liquid foodproduct,willalso

affectthesterilizationefficiency.FortypicalUHTpro-

cessingequipmentitcanbeassumedthatanaverage

of9–10decimalreductionscanbeachievedwithbac-

terialsporesgrowingatambienttemperatures.

Initial spore Spore load/l UHT decimal Spores/l Defect load /ml reduction in product rate

10000=104 107 9D 0.01 1:100

100=102 105 9D 0.0001 1:10000

More spores, more problemsInadditiontothefactorsmentionedabove,theeffi-

ciencyoftheUHTprocessisinfluencedbythespore

contentoftheliquidfood,i.e.thetotalsporeload,fed

intothefinalheat-treatmentsectionoftheUHTplant.

What is theeffectofdifferent initialspore loadson

end-product quality, assuming identical processing

conditions?Iftheproductisfilledinto1-litrepackages,

thedefectratecanbedeterminedfromtheinforma-

tiongiveninthetable.

Figure4illustratestheeffectofincreasedsterilization

temperatureoninitialsporeload,andtheeffectofdif-

ferentsporeloadsonthesametemperature.

Fig. 4. Increasing the temperature for a particular initial spore load achieves a quicker killing rate. Increasing the initial spore load for a particular temperature leads to a longer time required to achieve the same spore reduc-tion as for a lower spore load.

105

104

103

102

101

100

10-1

10-2

10-3

10-4

10-5

NUMBER OF SPORES

TIME

High temperature

Low temperature

Sterilizationefficiencycanbeexpressedasthenumberofdecimalreductionsinmicrobialpopulation.

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A mixture of remedies

The importanceof initial spore loadonsterilization

efficiency,andhenceend-productquality,isobvious.

Butwhatcanbedonetoimprovethesituation?The

most importantmethodsofachievingthisaregiven

belowandsummarizedinthetable.

Improved hygieneAhighconcentrationofsporesmayalreadybepresent

inmilkatthefarm,orbeintroducedintorawmaterials

asaresultofpoorpreprocessingroutines.Theanswer

hereistoimprovethelevelofhygienebothatthefarm

andtheprocessingplant.However,itisdifficulttocon-

trolwhathappensonafarmanditisnotalwayspos-

sibletochangemilksuppliers.

Spore reductionAgoodwayofimprovingtheend-productistoreduce

thesporecontentofrawmaterialsbeforetheliquid

foodisheat-treated.Processessuchasbactofugation

andmicrofiltrationarespeciallydesignedtogreatly

reducethesporecontentofmilk.

Effective heat treatmentAn increase in temperature and/or prolonging the

productholdingtimeduringheattreatmentwillresult

inamoreeffectivesterilizationprocess.Asaconse-

quence,thenumberofdecimalreductionswillincrease

andthedefectlevelwilldecrease.

Spore reduction remedies

Process Remedial action to reduce spore count Effectiveness

Milking Improvefarmhygiene Helpfulbuthardtocontrol

Changesuppliers

Preprocessing Improveplanthygiene Helpful

Sporereduction Bactofugation,microfiltration

orothermeasure Effective

UHTtreatment Increasetemperature

Prolongholdingtime Veryeffective

SporereductionandimprovedUHTtreatmentarethemosteffectivemeansofincreasingsterilizingefficiency.

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UHT treatment

Two principlesThere are two different UHT methods used in the

liquid food industry – direct and indirect heating.

Whichonetousedependsonanumberoffactorssuch

asdesiredendproductquality levelandproduction

economy.

Direct heatingWhenusingdirectheating,theproductisbroughtinto

directcontactwithhotsteamunderstrictlycontrolled

conditions.Thesterilizationtemperature(140°Cfor4

sec)israpidlyreachedandafterholding,thetempera-

tureisloweredbyflashcoolinginavacuumvessel.The

rapidheatingandcoolinggivesminimalheat-loadon

theproductwithahigh-qualityproductastheresult.

Indirect heatingInthismethodapartitionisplacedbetweentheprod-

uctandtheheatingorcoolingmedium.Theprinciple

isthatahotmediumisflowingononesideofthepar-

titionandtheproductontheother.Thepartition is

heatedupandtransferstheheatovertotheproduct

flow without any direct contact between the hot

mediumandtheproduct.Thesameprincipleapplies

tocooling.

Thisprocedureisdoneinwhatisreferredtoasaheat

exchanger.Whichonetochoosedependsonseveral

factors,e.g.productflowrate,physicalpropertiesof

the liquid, required running time, cleaning require-

mentsetc.

Thefollowingthreetypesofheatexchangersarethe

mostwidelyused:

• Plateheatexchangers(PHE)aremainlyused

forsmoothlowviscousliquidproducts.

• Tubularheatexchangers(THE)canalsohandle

productswithparticlesuptoacertainsizeand

offerlongerrunningtimes.Themaximumsize

ofparticlesdependsonthediameterofthetube.

• Scraped-surfaceheatexchangers(SSHE)are

speciallydesignedforheatingandcoolingof

viscous,stickyandlumpyproducts,withor

withoutparticles.

TheefficiencyandflexibilityofUHTprocessingarecon-

tinuouslybeing improved.One typeofUHTmodule

incorporates both direct and indirect heating with

tubularheatexchangerstoprovidedifferentcombina-

tionsofheattreatmentinonesystem.

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Microfiltration

Microfiltrationminimizesthebacterialandsporecontentofmilkinacost-effectiveway.

AcombinationofmembranefiltrationandUHTtreat-

mentisproventobethemosteffectivemethodof

increasingsterilizingefficiency.

Byusingamicrofiltrationmembrane,99.5-99.95%

ofbacteriaandsporesareeffectivelyremovedinacost-

efficientway.Thisisachievedbylettingtheliquidsub-

stancepassover amembraneathigh speedunder

pressureinaclosedsystem.

Themembranehasveryspecificproperties that

onlyallowthebacteriaandsporesofa“maximumsize”

to pass through the membrane. Since the size and

weightoftheunwantedbacteriaandsporesareknown,

thefilterused isconstructedwithappropriatepore

sizestoremovethesepartsfromtheliquidandletthe

smallerprotein,lactose,mineralandwatermolecules

passontofinalUHTtreatment.

Themainbenefitsofthisprocedurearethatthe

productwillbemorepurifiedbeforeUHTtreatment

andthefinalproductwillnotcontainanydeadspores.

Thisgivesanend-productwithhigherquality,better

tasteandlongershelf-life.

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Summary

• SporesposethebiggestchallengetoUHTprocessing

anddeterminethesterilizationparameters.

• Absolutesterilitycanneverbereachedorguaranteed.

• Eachlinkintheprocessingandpackagingchainmust

beequallystrong.

• Acceptancequalitylevels(AQL)formanintegralpart

ofacompany’squalitypolicy.

• Statisticalresultsareaffectedonlybythesamplingsize

andnotbythetotalnumberofunitsproduced.

• Moresampleswillimprovethedetectionlevel.

• Sterilizationefficiencycanbeexpressedasthenumber

ofdecimalreductionsinmicrobialpopulation.

• SporereductionandimprovedUHTtreatmentarethe

mosteffectivemeansofincreasingsterilizingefficiency.

��Tetra Pak, and ProTecTs whaT’s good are trademarks belonging to the Tetra Pak group.

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