Bacilli in Food-Eng

9/13/2015 Bacilliinfood

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Bacilliinfood

MICROFoodandEnvironmentalHygieneandcontrol

2011November21

MirjaSalkinojaSalonen

SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21

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CausingfoodpoisoningBacillusismainlyintwogroups:

Bacilluscereussensibarn(includesspeciesofB.cereussensustricto,B.thuringiensis,B.

mycoides,B.pseudomycoidesB.weihenstephanensis,B.anthracis)

and

Bacillussubtilisgroup(includingB.subtilis,B.amyloliquefaciens,B.mojavensisB.

atrophaeus).

Ofthese,theFinnishlawotherbiologicalriskgroups,...

17.10.2000ENOfficialJournaloftheEuropeanCommunitiesL262/21

DIRECTIVE2000/54/ECOFTHEEUROPEANPARLIAMENTANDOFTHECOUNCIL

of18September2000,



theprotectionofworkersfromtherisksrelatedtobiologicalagents

atwork

(SeventhreferredtoinArticle16ofDirective89/391/EEC,Article1

individualdirective

...Otherthan#1,isonlyB.anthracis(riskcategory3).InsomeEUcountries

B.cereusisariskgroup2,butthusFinland.B.anthracisdoesnotcause

foodpoisoning,butitssporescauseadangerousinfection

inhaledorthroughtheskin.


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Table10.IntrinsicandextrinsicpropertiesofB.cereusandB.subtlisgroupnoticeforgrowthinfoods

Bacterialbiochemicalpropertiesstarchdegradingactivity AbilitytohydrolyzestarchisacommonpropertyofB.cereusgroup

species 1 andBacillusisthemostnoticeamylolyticgenusforIndustrialproductionofdifferentstarchdegradingenzymes 2.MostemeticstrainsarenegativeforstarchHydrolysisalthoughtheyaremostfrequentlytoreportedinstarchyfoods.

proteaseactivity noticeformakingaminoacidsavailablefromproteinSubstrateslipolyticactivity noticeformakingfattyacidsavailablefromlipidSubstratessalttolerance Allowssurvivalinsaltstressenvironment.ImportantAlsoforthemotocross

protectionbetweenstresses.ForexampleinonestudyisB.cereus,saltprotectedagainsthydrogenperoxide,Whichprotectedagainstethanol,Whichprotectedagainstheatshock. 3

Intrinsicparametersofthefood 4pH B.cereusandB.subtilisgroupgrowatapHof5to9.5.Thepresenceof

saltaprwidenthepHrange.ToolowortoohighpHaltertheFunctioningofenzymesandthetransportofnutrientsintothecell.

Moisturecontent thewateractivity(a w)Ofmostfreshfoodsisabove0.99andB.subtilisforexamplecangrowatminimum0.95.Theminimuma wtoreportedforB.cereusgrowthis0.91to0.96infriedrice 5.Temperatureandnutrientsmightpermitgrowthatlowervaluesofa w:therangeofa wOverWhichgrowthoccursisgreatestattheoptimumtemperatureforgrowthandthepresenceofnutrientsincreasestherangeofa w OverWhichtheorganismscangrow.Alowa w increaseslengthofthelagphaseofgrowthanddecreasesthegrowthrate.

oxidationreductionPotentialEh

GenerallythemembersofthegenusBacilluspreferapositiveEhvalues(oxidized)forgrowth.

nutrientcontent includesthepresenceofwater,sourceofenergy(suchassugarsandaminoacids),sourceofnitrogen(likeaminoacids),vitaminsandrelatedgrowthfactorsandminerals.

AntimicrobialconstituentsPreservativesandfood

Includesessentialoilsinplants(likeEugenolincloves,alliciningarlic,thymolinsageandoregano,etc),Lysozymeineggsandmilk,Lactoferrininmilk,etc.FoodPreservativesaffectingsporeFORMERSincludenisin,benzoate,sorbate.

biologicalstructure thenaturalcoveringofsomefoodsprovidesprotectionagainsttheentryofspoilageorganisms(ietestaofseeds,shellsofeggs,ofnuts,theskin,etc).Presenceoforganicmatteraprprotectbacteriafromtheactionofdisinfectants.

Extrinsicparameters 4temperatureofstorage Generally,atemperaturelowerthan47Corhigherthan55Cwould

impedethegrowthofanymemberfromB.cereusorB.subtilisgroup.(Table1andTable6).

relativehumidityofenvironment

Whenthea w ofafoodisforexample0.6,itisnoticetostoreitunderconditionsThatPreventthefoodabsorbingmoisture.

thepresenceandconcentrationofgases

CO 2 isthemostnoticeatmosphericgasThatisusedtocontrolmicroorganismsinfoods.Bacillussp.areamongthemostsensitivemicroorganismstoCO 2 relativetomodifiedatmospherepackaging


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B.cereusisacommonbacterialeverywhereintheenvironment,especiallyplants(including

crops)intheroot(rhizosphere)andalsoplantendophyte.B.cereusisnot

harmfultoplantsandisfoundintubersandgreenparts,includingwoody.

Plantsarenotcontainedsterile(withtheexceptionoffruit)asanimals.Plant

watercanalsfoundinbacteriathatshallcomeintosoilwaterwiththerootsystem.



Table2.EnvironmentalnichesofB.cereusgroup

Species soila water b food c mammaliangut d

insectgut e

earthwormgut f

plantrhizosphereorendophyteB.cereussensustricto+ + + +s + +

B.anthracis + + +B.thurigiensis + + + +s + +B.mycoides + + + + + +B.pseudomycoides + + +B.weihenstephanensis+ + + +aVilainetal.,2006Jensenetal.,2003vonStettenetal.,1999Thorsenetal.,2006

bstensviketetal,2004..Jensenetal,2003

cAstraightforwardArnesenetal.,2008Povetal.,2003Kajikazawaetal.,2007Frederiksenetal.

2006Hansonetal.,2005

dWilksetal.,2007Jensenetal.,2003

eJensenetal.,2003Cooketal.,2007

f Jensenetal.,2003

gJensenetal.,2003Okunishietal.,2005SaileandKoehler,2006Jafraetal.,2006

stransient


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Table2.ThepublishedcompletegenomesequencesofB.cereusstrains.

BacilluscereusstrainSize(bp)Numberof

genes Links Reference

ATCC10987 5224283 5603 NCBIRefseqNC003909Raskoetal.,2004ATCC14579 5411809 5234 NCBIRefseqNC004722Ivanovaetal.,2003

ZK 5300915 5134 TIGR,Tax288681 Hanetal.,2006NVH39198 4087024 4165 NCBIRefseqNC009674 Unpublished

E33L 5300915 5269 NCBIRefseqNC006274 Hanetal.,2006

Bacilluscereusisthe2000'sintensiveresearchastrack,becausethe

ruokamyrkyttjnhasincreasedtheimportanceoflongtermfoodlogisticschains

timeandnumberofmoveseatingcomfortfoods(heatandchill"kitchens").B.

cereuPRINCIsporesarehighlyresistantkuumuttaandaccumulateinthefood

productionprocesses,withtheheatingstep(=rivalsarereduced).

Formorethan10B.theentiregenomescereusstrainissequenced.

B.cereusandB.somestockssubtilisgroupproducingThermotolerantpeptiditoksiineja,

whicharehighlytoxictohumans,andhavecausedkuoleemankinled

foodpoisoning.Thesetoxinsremaininthefood,eventhoughitisbyheating

destroyedalllivingbacteria.


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PhenotypictraitsusefultodifferentiatewithinthemembersofB.cereusgroup

Species haemolysismotilityparasporalinclusion

susceptibilitytopenicillin

colonymorphology

growthtemperature(C)

lysisbygammaphageB.cereussensustricto+ + white 455

B.anthracis + white 1540 +B.thurigiensis + + + white/gray 1045 B.mycoides + rhizoid 1040 B.pseudomycoides* + + white/cream,

rhizoidal1540

B.weihenstephanensis+ + white 738

*DistinguishedfromB.mycoidesbydifferencesinwholecellfattyacidcomposition12:0isoand13:0

anteisolevelsandfromB.cereusbydifferencesin12:0iso,12:0,15:0isoand16:0(Nakamura,1998).

4of5strainsofB.mycoideswereresistantand5of6strainsofB.pseudomycoideswereSusceptible

DatafromLunaetal.,2007

CompiledfromGranum,2007Gordon,1973Lechneret.al.,1998Nakamura,1998vonStettenetal.

1999Roberts,etal.,1996b.

Bacilluscereusisaveryheterogeneousgroup,eventhoughitsmembersseparationofthe

morphologicalorbiochemicalcharacteristicsisdifficult.Thistablecontainsteststhatcan

usedtodistinguishpathogenic(sub)speciesfromtheother.

GammafaagiapidetnasspecificB.anthracisspeciesof,butgammafaagilleherkkiB.cereusstrains

ismm.foundinthedairies'koticereuksista".TheyarenotexpectedtoproducehavattuB.anthracis

characterizedtoxins(lethaltoxinandedematoxin).B.anthracisthespeciesmostresemble

oksetustoksiinia(cereulide)producedbytheB.cereusstrainsthatarenegative(oralmost

negative)haemolysisasB.anthracis.

B.mycoides,andB.pseudomycoidesareknowntohavecausedfoodpoisoning .


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BiochemicaltestsusedintheidentificationofBacilli:

HemolysisTyrosinase:positiveontheleft,ontheright

negative




Lesitinaasitesti,Thepositiveleft,right,anegativeresult.Thetestmeasuresmunankeltuaisagarinsisltmnlecithindegradationofglycerolandfattyacids.Fattyacids

precipitatefromthereactionzone

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B.cereusATCC14579:TEMimage

negatiivivrjtyistspores.Ranad

Shaheen,2008.Bar=1microns

B.cereusF4810/72:TEMimagespores

thinsection(MariaAndersson)

B.cereussporesofthebacteriumresistanttomany

heating.Particularlydurableare

cereulide(oksetustoksiini)producing

Sporesofstocks.


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ScanningElectronMicroscope

(SEM)imagesB.cereusbacterium



sporesadheredtothesteelplatesurface.Theleftimageshows

steelsurfacegranularity(granulelimits).

RanadShaheen,2008.

B.cereus(sensulato)isthesurfaceofthebacterialspores

hydrophobic,i.e.waterrepellent.Thereuponit

sticksoutofthewaterorfoodhydrophobic

surfaces,especiallyplasticsandsteel.

Adheredtothesurfaceofthesporeisdesinfioitavuus

poorbecausethesurfaceoftheadheredsporesareresistantto

heatandchemicaldisinfectionmore

thesporessuspendedintheliquidphase.SalkinojaSalonen,M.,Bacillus

foodstuffs2011November21

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CharacteristicoftheTablecereulideProducingB.cereussporestestedinpaperIIIandIV

Cereulide

ProducingB.cereus

Cereulide

nonProducingB.cereus

Numberof

cereulideProducingstrainsOutofThosetested

Resistanttoheatat90CD(min)value 370 90 17/81

(min)value 25120 2530 17/81

log10Viabilitylossafter120min

at90C

1 4 17/81

Log 10 (N 0/N)germinationat7

Cafter7days

0.2 1.82 17/81

Log 10 (N 0/N)germinationat30

Cafter50min

1.8 1.82.5 17/81

Resistanttohotwashingwith:

Alkaline(1%NaOH+75C,pH13.1)

RangeD(min)initialRangeoflogkill15min

11.413.61.42.5

1.1670.15

3/203/20

Acid(0.9%,65C,HNO3)RangeD(min)initialRangeoflogkill15min

3.3

3.5

1.325

0.45.9

1/6

1/6

Cereulide(B.cereus

producedbythebacterium

oksetustoksiini)

producingstrains

differfromotherB.

cereusstrains

inthattheirspores

resistantheating

IDandanalkalinewash

multiply

morethan

"Ordinary"antidiarrheal

diseasescausedbyB.

cereusstrains.

Particularlydifficultis

theirhightolerance

hotalkalinewash

andnitricacid

flushing,whichare

aremm.dairies

usedequipment

Cleaningmethods

Source:RanadShaheen,PhD,UniversityofHelsinki,DissBiocentr.Molec.UnivHelsigiensisinVik34/2009


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10 7

10 8

10 9Cfu/ml Whenthefoodproductispasteurized(eg.Milk,

72C)suchasB.cereusspores,exceptthat

retaintheirviability,pikakynnistvt

germination,sothatthevegetativeB.cereus

theconcentrationofbacteriainthefoodrises

eg.10100times.Ifnot

heatedwould,butthefood



0 1 2 3 4 5 6 7 8 9 1010 2

10 3

10 4

10 5

10 6

Cookingtime,minutes


bemaintainedatalowertemperature,

sporegermination(germination=)starts

onlyslowly:forexample,7C.inmaytake

aweekbeforeeven10%ofthosepresent

getssporestogerminate.FrozenPastry(eg.

Karelianpasties)omapaisto(250 C)in

initiatesporegermination,ietheproduct

containedinB.cereucanquicklygs

multiplyandcausefoodpoisoning,

unlessthepietoeatsoon.

However,pasteurizationisoftennecessary

otherfoodpollutersandhumanhealthhazards

causingthedestructionofbacteria(eg.

mycobacteria,listeria,salmonella).

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Table4.PhenotypicandgenotypicpropertiestoreportedasspecificforthecereulideProducing

strainsofB.cereus.

Properties Reference

Phenotypictraits

weakhaemolysis Anderssonetal.,2004

inabilitytostarchhydrolysis Shinagawaetal.,1979

salicindecompositionnegative Shinagawaetal.,1993

H1predominanceofserovar Agataetal.,1996

lackoftheHaemolyticenterotoxin

Production

Agataetal.,1996

Genotypictraitsspecificribopattern Pirttijrvietal.,1999

specificRAPDprofiles EhlingSchulzetal.,2005a

specificSDSPAGEprofilesexoproteinEhlingSchulzetal.,2005a,

Identical16SrRNAgenesequences EhlingSchulzetal.,2005a

IdenticalMLSTsequencetype EhlingSchulzetal.,2005a

IdenticalFTIRspectra EhlingSchulzetal.,2005aSalkinojaSalonen,M.,Bacillusfoodstuffs2011November21

Bacilluscereusproducesmanydifferenttypesofproteintoxin.Theyareproducedby

fisheatenfoodgastrointestinalbeenvegetativebacteria,

Sporesandtheirdescendants.Startingthisway,diarrhealdiseaseisinfectiousand,therefore,

diseases(suchassalmonellosis,campylobacteriosis,yersiniosis,etc.),but

usuallyofshortduration.

Cereulidethatis,

oksetustoksiinia,

producingB.cereus

positionsdiverge

ripulitoksisistainthat

theyproducethetoxin

formedalreadyinourfoodand

causeeatenalmost

immediatepoisoning

(toksikoosin).Cereulide

producingstrains

differinanumber

physiologicaland

genetic

properties

otherB.

cereusstrains.They

aremm.usually

hemolysisare

todegradestarch,and

growhigher

attemperaturesotherthan

B.cereugs(upto+

Until52C)

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Starch

hydrolyysikyky

(amylolyyttisyys)is

generallypositive,aswellas

B.cereusgroupthatB.

subtilisgroup

bacteria.

Salicin(orthohydroxybenzylalcohol)isusedasthetestsubstancebetaglucanase

fortestingtheactivity.Betaglukanaasiwhereintheenzymehydrolyzesanetherbond

formedsalisyylialkoholia.

Cereulideproducingstrainsareusuallybothtrkkelysnegatiivisiathatsalisiininegatiivisia,

So,unlikeotherB.cereus.

salicin

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Molecularstructure Cyclicdodecadepsipeptide,1152g/mol

Synthesis Nonribosomal

Sensoryproperties ColourLess,odorless

Octanolwatercoefficient LogKow6.0

Heatstability Nolossofactivityuponcookingorautoclaving

pHstability Stablebetween211

Blacklipidmembrane Potassiumionophore

Isolatedratlivermitochondria CatalysesafluxofK+ions

Modeofactioncell Depolarizedmitochondrialmembrane

ofboarspermcells

ofNKcells

ofneuralcells

ofisolatedratlivermitochondria

Bacilluscereusoksetustoksiini:cereulidemoleculeproperties.Cereulideinwater

insoluble,lipophilicmolecule,whichcarriespotassiumjonejathroughbiologicalmembranes,so

troublemakerse.g.humanbodypotassiumhomeostasis.Itisalsoamitochondrialpoison.Cereulide

withstandwithoutlosingitsactivityautoclaving,acidandalkalitreatment,drying.

Cereulidestructure1152g/molpeptiditoksiini:

cyclo(OValLValODLeuDAla)3




Page15


Spermmitochondriaarelocatedsiittihnnn(=flagellin),thebasepart.Ofthespermheaddoesnothaveany

mitochonidria.Thisuniquearchitectureallowsthecellmitochondrialdamageeasy

Detectionbylightmicroscopy,e.g.,JC1stainedcellswithafluorogenic.FigureswimJC1

colored,healthypigspermatozoawithhead(=lowmembranepotential)fluorescesgreenandthetail

thebaseofthemitochondriaorange.

Exposedcereulidepig

spermJC1colored.

Ispartofthemitochondria

lost

andtheelectricchargeofthesperm

hyperpolarisoitunutend.

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Table7.EnvironmentalnichestoreportedforthemembersofB.subtilisgroup

Species soil a water b food c mammaliangut d

insectgut e

Industrialfermentations f

intherhizosphereorasendophyteofplants g

B.subtilisssp.subtilis + + + + + + +B.amyloliquefaciens, + + +B.atrophaeus +B.licheniformis + + + + + +B.mojavensis + + +B.pumilus + + + +B.subtilisssp.spizizenii +B.vallismortis + + +B.sonorensis + +a PriestF.1989Nakamuraetal.,1999Roberts,etal.,1996aNakamura1989Roberts,etal.,1994

Palmisanoetal.,2001

b PriestF.1989stensviketal,2004..Jeongetal,2007

BacillussubtilisbacterialspeciesgrouplivinginthesameenvironmentsasB.cereus.



c PriestF.1989Frometal,2005..Sorokulovaetal,2003

d Tametal.,2006Leseryou.al.,2007

eKnigH.2006

fOchiaietal.,2007PriestF.1989

g Idrissetal.,2002BaconandHinton,2001Manoetal.,2006Parketal.,2007


Page17


Amyloseisahydrophobicpeptide,1197Da,composedofleucine,proline,

serine,asparticacid,glutamicacidandtyrosine.Itisidentified_byandquantitated

basedonitsmolecularions(1197m/z)

AmyloseFormedcationselectivechannelsinlipidmembranes,witha

selectivityK +Na +Ca 2+ of26:15:3.5The

ExposuretoAmylosedepolarizedthetransmembranepotentialsofmitochondria

( m)andoftheplasmamembrane( p)ofprimaryboarsperm,felinelung,human

NKcellsandcelllines(lungCalu3,neuralPaju)uponwithin20minofexposure

Bacillusamyloliquefaciensoffoodbornebacterialtoxin

Amylose.

Amyloseproducespotassium,sodiumandcalciumjonejapermeabletroublemakerchannels

mammaliancells.Innateimmunecells(macrophages)Amylose

sytokiinimyrskynstart,ifpresent,isatthesametimesomeofthemicrobialcell

componentssuchase.g.lipopolysaccharide(LPS).

Page18

Bacillussubtilisheatstabletoxinsproducedbythegroup:

Testcellsperm(swine)



Humantestcell

colon

Adenocarcinoma

(Caco2)


Page19


IonophoricactivityofAmylosefromBacillusamyloliquefaciens

measuredbyblacklipidmembranes(BLM)

0.1 1101

100

101

102

103

104

105

106

CNonducsance,nS/cm2

Concentration,micrg/ml

CaCI2

NaCl

0.0 0.2 0.4 0.6

0

5

10

15

20

25

30

mV

thelogoftheconcentrationratio

KCI

logconcnofsubstance

alogofelectricconductivity

ofthelipidmembrane

Voltageacross

themembrane

concentrationratioofionsTefloncuvettewith50um 2

membranecoveredwindow

ionchannels

Permeabilityratio

ofchannelsfor

cationsover

anions:

K + /Cl >26

Na + /Cl =15

Ca 2+ /Cl =3.5

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JC1stainingRevealstheeffectsoftheHPLCpurified




AmyloseonboarspermcellsandhumancolonepithelialcelllineCaco2

Boarsperm Caco2

StructureofAmylosewasRevealedby

MS/MSandH1NMR

Amylosedepolarisedthemitochondrialand

plasmamembraneelectricpotentials:

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Amyloseisadangersignaltothehuman

macrophages

Dangersignal

LPS

Source:StiinaRasimus&




SampsaMatikainen

Page22


Toxicfoodpoisoningmeansthataman

makingsicktoxinwasalreadypresentinthefood.

Livemicrobesisnotnecessarilyfoundinfood(can

bekilledbyheating).Toxic

ruokamyrkytykstenmostcommoncausesare

fromtheEU/EFSAreport(theks.oikeallalist).

Otherfoodpoisoningsaremainlyinfections,ie,

microbial(eg.Bacilluscereus)hasenteredthefoodor

beveragewithgastrointestinalbegunand

theremultiplyandproducetoxinsor

virulencefactorsofwhichisthehumanbody

disadvantage.

TheEUandEFSA(EuropeanFoodSafetyAgency)compiledbyamassivedatabasefoodpoisoningsEuropeshowstheBacilli,inparticularBacilluscereup,theroleisEurope,significanttoxicfoodpoisoningacause.

Page23

Bacilli

and

standards

Bacilluscereus

determination

foodis

ThetwoISO

Standard:SFS

ENISO7932

(nextdoor)and

ISOEN21871:

MicrobiologyoffoodandanimalfeedingstuffshorixzontalMethodforthe




determinationofpresumptiveBacilluscereus.MostProbableNumber(MPN)techniqueanddetectionmethod.Colonycountingtechnique

at30C.2005

Thus,foodshavenotbeenthresholdsB.cereusconcentration.Specialgroups

(infants,immunocompromised)thepreparationoffood

processhygienemustbeofsuchqualitythattheB.cereusspores

contentdoesnotexceed50m=(satisfactory),M=500(acceptable)cfu/g.(EU

CommissionRegulationNo1441/2007.Off.J.Eur.Lunion,322:1229).

Page24

EscherichiacoliBacillussubtilisspores

1

0.1

0.01

10

pH4

pH7.9

Glutaaridialdehydi,0.0044%Glutaaridialdehydi2%

GTAalsoinactivatesvirusesandspores.Dangerousbyinhalation,allergenic,tosensitize.

OPAismoreeffectiveformycobacteriaandfatsolublesubstancesinanenvironmentlikeGTA,

apopulardisinfectantinhospitals,especiallyequipmentmaintenance,aneffectivemicrobicideandsporisidiand

moreresistanttoorganicdirtloadthanmanyothermaterials.Itdoesnotattackrubber,metals,

plastics,instrumentsorotherinstruments.

GTAishighlytoxictohumans,skincontactandinhalation.

OPAisaseffectivemicrobicidethantheGTA,butweakersporisidi.Itissafertotheuser

asGTA(evaporatesless).

60 120 180min


Bacillussporesaredifficulttodisinfect.Sporesbitesglutaricandopalaldehydes.Theeffectdependson

pHandcontacttimewillbelong.Duetotoxicresiduesofthesesubstancesarenotsuitable

ofthefoodprocessingenvironment.

Page25

DisinfectantsBacillusspores.

Chart2.2.4.aBacillussubtilissporesPRINCI

hydrogenperoxidetolerance.

AccordingtoW.Paulus(1993)

H 2O 2 Pit% Survival%,and

exposuretime(min)

2 30 60

Chart2.2.4b.Sporeproducingpowerofperaceticacidbacteria.Thefiguresrepresenttheminimumkilltimenuutteina.Bacterialinitialconsistencyof10 7 cfumlBosher1987,andPaulus1993basedonthefacts.BacterialSpecies Ksittelyl

mptilaPeraceticacidcontentinmgl

C 200

Bacilluscereus 5 >6020 >60

40 40




3 85 22 2.1

10 35 0.0027 0

15 22 0,0022 0

Bacillussubtilis 5 >6020 1040 10

Clostridiumperfringens

5 >60

20 >60

40 2.5

CereuPRINCIBacillussporesaremoredurablethanBacillussubtilis.Foodand

thepackagingindustryusedforthedisinfectionofsporesofhydrogenperoxide(bestpowerwill

heated)andperaceticacid,whichissoldinaceticacid,peraceticacidand

hydrogenperoxidetasapainoseoksena.Ofthese,donotleaveanytoxicresiduestotheenvironment.

CereulideproducingstrainsofB.cereussporesstocksareevenmoredurablethanotherB.

cereusten.

Page26


StandardmethodsforsporisidisenpowermeasurementI.Thesporesfordisinfection

soldmaterial

sporisidinen(=spores

Lethal)power

measurable

standardized

methods.

Elintarvikeymprists

Theweatheruse

theintendedsubstances

powerstudy

appended

standards,

oftestis

accordingtothestandard

B.usesubtilis

DSM347(=ATCC6633)

orB.cereus

CIP105151(=ATCC

12826)spores.

Surfacedisinfection

protocolsfor

sporisideilleis

separatemethods.

Page27

Standardmethodsforsporisidisenpowermeasurement2.




Researchingthepowerofsubstancesintendedforhumanandanimalskindisinfectionisnotsafety

reasonsusedBacillussporesexperimentaleliin.AccordingtoEN1499:1997,isusedtoE.coliK12

position(=tamedversionofthehumangutbacteriumE.coli"to).Itdoesnotproducespores.Themostcommonlyused

skindisinfectant,70%ethanol,spikedwithorwithoutalowconcentrationofchlorhexidine.Skin

isinvolvedintheprotectionofglycerol.Sporisidistakinthistreatmentiseffectiveifandwhentheskinisnot

aftertreatment,rinsewithwater,butlettheethanolevaporate.

Sporisidinenpowerbyanumericalvaluelog[N /N],whereN istheamountofsporeswithgrowth

incontrol(=lumeksitelty,orusedasdisinfectantneutralizinginactivated)andN

istheamountofsanitizingprocessingpassesthroughthesample.OutputNumber=0means,therefore,

thesituationinwhichbothhadthesamenumberofviablesporeinactivationisnotatapahtnunut.

Sporisidinenpowerofthechemicalishighlydependentonthedurationoftheprocessingoperation,temperature,pH

,theactivesubstanceconcentration,andinthedegreeofcontamination(dirtconsumingdisinfectionefficacy).

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Bacilli in Food-Eng