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Page1
Bacilliinfood
MICROFoodandEnvironmentalHygieneandcontrol
2011November21
MirjaSalkinojaSalonen
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
Page2
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,
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theprotectionofworkersfromtherisksrelatedtobiologicalagents
atwork
(SeventhreferredtoinArticle16ofDirective89/391/EEC,Article1
individualdirective
...Otherthan#1,isonlyB.anthracis(riskcategory3).InsomeEUcountries
B.cereusisariskgroup2,butthusFinland.B.anthracisdoesnotcause
foodpoisoning,butitssporescauseadangerousinfection
inhaledorthroughtheskin.
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
Page3
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
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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B.cereusisacommonbacterialeverywhereintheenvironment,especiallyplants(including
crops)intheroot(rhizosphere)andalsoplantendophyte.B.cereusisnot
harmfultoplantsandisfoundintubersandgreenparts,includingwoody.
Plantsarenotcontainedsterile(withtheexceptionoffruit)asanimals.Plant
watercanalsfoundinbacteriathatshallcomeintosoilwaterwiththerootsystem.
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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
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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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.
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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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 .
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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BiochemicaltestsusedintheidentificationofBacilli:
HemolysisTyrosinase:positiveontheleft,ontheright
negative
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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.
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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ScanningElectronMicroscope
(SEM)imagesB.cereusbacterium
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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
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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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
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0 1 2 3 4 5 6 7 8 9 1010 2
10 3
10 4
10 5
10 6
Cookingtime,minutes
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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).
Page12
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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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
Starch
hydrolyysikyky
(amylolyyttisyys)is
generallypositive,aswellas
B.cereusgroupthatB.
subtilisgroup
bacteria.
Salicin(orthohydroxybenzylalcohol)isusedasthetestsubstancebetaglucanase
fortestingtheactivity.Betaglukanaasiwhereintheenzymehydrolyzesanetherbond
formedsalisyylialkoholia.
Cereulideproducingstrainsareusuallybothtrkkelysnegatiivisiathatsalisiininegatiivisia,
So,unlikeotherB.cereus.
salicin
Page14
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
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
Spermmitochondriaarelocatedsiittihnnn(=flagellin),thebasepart.Ofthespermheaddoesnothaveany
mitochonidria.Thisuniquearchitectureallowsthecellmitochondrialdamageeasy
Detectionbylightmicroscopy,e.g.,JC1stainedcellswithafluorogenic.FigureswimJC1
colored,healthypigspermatozoawithhead(=lowmembranepotential)fluorescesgreenandthetail
thebaseofthemitochondriaorange.
Exposedcereulidepig
spermJC1colored.
Ispartofthemitochondria
lost
andtheelectricchargeofthesperm
hyperpolarisoitunutend.
Page16
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.
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c PriestF.1989Frometal,2005..Sorokulovaetal,2003
d Tametal.,2006Leseryou.al.,2007
eKnigH.2006
fOchiaietal.,2007PriestF.1989
g Idrissetal.,2002BaconandHinton,2001Manoetal.,2006Parketal.,2007
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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)
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Humantestcell
colon
Adenocarcinoma
(Caco2)
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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
Page20
JC1stainingRevealstheeffectsoftheHPLCpurified
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
AmyloseonboarspermcellsandhumancolonepithelialcelllineCaco2
Boarsperm Caco2
StructureofAmylosewasRevealedby
MS/MSandH1NMR
Amylosedepolarisedthemitochondrialand
plasmamembraneelectricpotentials:
Page21
Amyloseisadangersignaltothehuman
macrophages
Dangersignal
LPS
Source:StiinaRasimus&
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
SampsaMatikainen
Page22
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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
SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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.
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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.
9/13/2015 Bacilliinfood
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SalkinojaSalonen,M.,Bacillusfoodstuffs2011November21
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).