Post on 04-Aug-2020
NATURALANTIMICROBIALSTheAbilitytoMaintainSkin’sMicrofloraBalancevs.theDamagingEffectsof
TraditionalSyntheticPreservatives
OUTLINE
I. ImportanceofPreservation
II. TraditionalPreservatives
III. AlternativeAntimicrobials
IV. BacterialSymbiosis:Resistant,Transient,andCommensal
V. IdealBalance:Eliminating“Bad”&Promoting“Good”Bacteria
VI. AlternativesinAction
ImportanceofPreservation
Step1- Theusualsuspects
1.BacteriaandFungus(YeastandMold)I.Gram-Positive– crystalviolet– StaphII.GramNegative– fuchsine– PseudomonasIII.Pathogens
2.HouseOrganisms
3.UsualsourcesofcontaminationI.AirborneII.RawMaterialsIII.Lines/Valves/Pumps/Tanks
ImportanceofPreservation
Step2- Produceacleanproduct
1.Goodmanufacturingpractice(GMP)I.ColdorhotasepticfillingII.Clean-roomtechnology
2.Hygiene-compliantpackaging3.Emulsionform4.Wateractivity5.pHcontrol6.Multifunctionalingredientswithbiostaticeffect7.Remediation– Thermal– Gammaradiation
SchuelkeMeyer- Useofmultifunctionalstoachieveself-preservingsystems
PreservationBasics
1. APC(AerobicPlateCounts)
2. PET(PreservativeEfficacyTest)aka“ChallengeTest
3. PreservationChallengesI. CompatibilityII. NaturalGuidelinesIII. GlobalRegulationsIV. EfficacyV. Change
APC(AerobicPlateCounts)
1. PicksupBacteria,Yeast,andMold
2. TrypticSoyAgarwithlecithinandpolysorbate80temperedat45°C(TSA)SabouraudDextroseAgartemperedat45°C(SDA)
3. 2daysforbacteria5daysforfungus
PET(PreservativeEfficacyTest)aka“ChallengeTest”
1.PCPCmethodM-1.2.PCPCmethodM-3orUSP<51>ChallengeS.aureus,E.coli,P.aeruginosa,C.albicans,andA.brasiliensis
3.PassCriteria>99.999%%at7daysforbacteria>90.0%at7daysforfungus
4.AcceptableLevels
•Babyproducts- nomorethan10²CFU/gram•Eyeareaproducts- nomorethan10²CFU/gram•Allotherproducts- nomorethan10³CFU/gram
TraditionalPreservatives
I. US– EPApesticideregistrationII. Europe– AnnexVoftheCosmeticActIII. Asia– countrybycountryadministeredbytheequivalentoftheFDA
AlternativeAntimicrobials
I. Botanicals–WillowBark/Honeysuckle/WoodExtractsII. GlycolsIII. Organicacids/EstersIV. PeptidesV. Packaging
IMPORTANCEOFPRESERVATION
TheSkin
• Thebody’slargestorganandonethatisconstantlyexposedtotheenvironment
• Itprotectsthebodyfrompathogenswhilesustainingmicroorganismsthatinfluencehumanhealthanddisease.
• Anideallocationforthecontrolledgrowthofbacteria
• Consistsofvariouscutaneousantimicrobialdefencemechanisms:
• StratumCorneum• StratumCorneumLipids• ProductionofLysozyme• Acidity• Defensins• CommensalBacteria
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
SkinMicrobiome
• Theskinisanecosystem,harboringmicrobialcommunitiesthatliveinarangeofphysiologicallyandtopographicallydistinctniches
• Bacterialgrowthontheskinincludes:• Resident&TransientPathogenicBacteria- capableofinvadingandcausingharm• CommensalBacteria- protectsthehostfromthesepathogens
• A2009NIHmicrobiomestudyanalyzedribosomalRNAgenesequencesobtainedfrom20distinctskinsitesofhealthyhumans
• Revealedthatphysiologicallycomparablesitesharborsimilarbacterialcommunities
• Thecomplexityandstabilityofthemicrobialcommunityaredependentonthespecificcharacteristicsoftheskinsiteandextrinsicenvironmentalfactors
• Examinedthemicrobialinterdependenciesrequiredtomaintainhealthyskin
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
SkinMicrobiome
SkinMicrobiome
• Recentresearchhasbeguntodocumenthowskincommensalsinteractwithoneanother,withpathogenicmicrobes,andwithhumancells.Examples:
• Staphylococcusepidermidissecretesantimicrobialsubstancesthathelpfightpathogenicinvaders
• P.acnesusestheskin’slipidstogenerateshort-chainfattyacidsthatcansimilarlywardoffmicrobialthreats
• “Ithinkanythingwecandotorestoremorebalanceormoreappropriatemicrobecompositionintheskin,asinallthedifferenttissues,isextremelyimportant.”
- YasmineBelkaidoftheNationalInstituteofAllergyandInfectiousDisease
• Theseandotherskinmicrobescanimpactthelocalmolecularenvironment,andmaybeabletoalterthebehaviorofhumanimmunecells
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
IMPORTANCEOFPRESERVATION
CosmeticPreservatives
• Theproperuseofpreservativesandbiocidesaretopreventmicrobialcontaminationwithoutalteringtheskinmicrobiome
• GramnegativeandGrampositivebacteria,yeasts,andmoldshaveallbeenfoundtogrowinvariouscosmeticproducts
• Yet,someofthesemicrobesarealsoresidentcommensalbacteriaontheskin
• Activebiocidesmusteliminatethebad,yetpromotethegoodbacteria
• Sometraditionalpreservativeseffectiveatreducingcosmeticcontamination:• Parabens• Formaldehydedonors• Halogenatedcompounds
• Butaretheysafe?
RoleoftheHDAC
• HistoneDeacetylaces(HDAC’s)areaclassofenzymesexpressedinskincells
• Theseenzymeshelpmaintainhealthyskin bybalancingacetylationactivitiesofhistoneacetyltransferasesonchromatinremodelingandalsoplayessentialrolesinregulatinggenetranscription
• Canberesponsivetomanydifferentenvironmentalsignals
• Specifically,HDAC3whichisthemostprominentlyexpressedhistonedeacetylaseinN-TERThumankeratinocytecells,hasvariousinflammatoryandmetabolicroles
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
HDAC3• Researchhasfound thatHDAC3isakey
mediatorinmaintainingtheoverallintegrityandfunctionofhumanorganssuchastheintestinesandskin,whileinthepresenceofcommensal“friendly”bacteria
• HDAC3alsoplaysakeyroleintherelationshipbetweenmicrobiotaandinflammation
• Arecentstudyfound thatthereweremajordifferencesbetweenthemicrobialpopulationdiversitywithinnormalmicecomparedtomicedeficientinHDAC3
• Somebacterialspecieswereactuallyover-populatedwhenHDAC3wasreduced
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
HDAC3
• ResearchprovedthatthereisafundamentalchangeintherelationshipbetweencommensalbacteriaandtheirmammalianhostsfollowingdeletionofHDAC3
• HDAC3notonlyinfluencesthebacterialpopulation,butthebacteriainturninfluencecells’behaviourasaresult,whichincludesgeneexpression
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
RoleofHDAC
• A2012studyfromtheJournalofCellScienceinvestigatedHDACproteinlevelsandtotalactivitydependenceonArylHydrocarbonReceptorNuclearTranslocator(ARNT)inN-TERTepidermalkeratinocytescells
• AnHDACactivityassaywasemployed
• HDAC3appearedtobethemostprominentlyexpressedhistonedeacetylaseinthesecells
• Attheproteinlevel,depletionofARNTleadtoasignificantincreaseofHDAC3
• InARNT-depletedHaCaTcells,therewasalsoaprominentincreaseinHDAC3proteinlevels
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
RoleofHDAC
• TheJournalofBiologicalChemistrypublishedastudy investigatingHDAC3mediationofskininflammation
• TheroleofHDAC3inallergicskininflammationprimarilyremainsunknown
• TheirresultsindicatethatHDAC3interactswithFcεRIandregulatesexpressionofMCP1throughSp1andc-Juntomediateallergicskininflammation
• ConcludedthatHDAC3mediatesallergicskininflammationinrelationwithangiogenesisbyregulatingMCP1
• HDAC3servesasatargetfordevelopmentofallergytherapeutics
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
RoleofHDAC
• HDACexpressionwithinmultipletissuesystemssuchasthedigestivetractandskin,isanintegralfactorinorganhealthandfunction
• Thisenzymeisextremelysensitivetoenvironmentalandintrinsicfactorssuchaspreservativesandbiocides,i.e.:
• Triclosan• Parabens
• ExpressionofHDACisanessentialcomponentofhowweregulatetherelationshipbetweencommensalbacteriaandcellfunction tomaintainhealthyskin
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
RoleofHDAC
• WhenHDACisalteredorreducedthentheskin’scommensalbacterial,whichprotectsagainstunwantedmicrobes,isnolongeraseffective
• OurresearchhasshownmosttraditionalbiocidesdecreaseHDACexpressioninhumankeratinocytes
• Thisleadstoacompromisedimmunedefencesystemandreducedskinhealth
• Whatifbiocidescouldonlyeliminatethe“bad”withoutalteringthe“good”commensalmicrobes,whilestillnotaffectingHDACactivity?
• Couldtraditionalpreservativesdothisordonaturalantimicrobialshold thekey?
BACTERIALSYMBIOSIS:RESISTANT,TRANSIENT&COMMENSAL
TRADITIONALBIOCIDES
Triclosan
• Triclosanisabactericidalbroad-spectrumagentdevelopedover40yearsagoandfirstintroducedasasurgicalscrub
• Primarilyusedasatopicalbiocidemoresothanacosmeticproductpreservative,suchasparabens
• Bothbiocidesandpreservativesaffecttheskinmicrobiome
• Overthelast20yearsitsusehasgrownrapidlyinpersonalcareproductsincludingsoap,handsanitizer,cosmetics,andtoothpaste,aswellashouseholdproductssuchasodor-fighting socksandgerm-resistantsponges,kitchenware,andbedding
TRADITIONALBIOCIDES
Triclosan
• Cancausedisruptionofbacterialcellwallsinnonspecifictargets
• DecreasesHDACexpressioninskinkeratinocytes
• Resultsindisturbanceoftheskin’smicroflorabalance• Pathogenicandcommensalbacteriaarekilled• Skinleftdefenselessagainstnewdestructivemicroorganisms
• Canalsocausedangerousantimicrobialresistancetovitalmedicines• Growingthreattohealthcareasawhole
• Mayenhancetheproductionofchloroform,whichisclassifiedbytheEPAasaprobablehumancarcinogen.• A2007studyillustratedthat,undersomecircumstances,triclosantriggered
productionofchloroforminamountsupto40%higherthanbackgroundlevelsinchlorine-treatedtapwater
NATURALANTIMICROBIALS
NewFormulationApproach
• Somenaturalantimicrobialsareequipped tokillpathogenicbacteriawhilestillmaintainingavigorouscommensalmicrofloraontheskin
• OthernaturalantimicrobialswillnothaveanegativeorinhibitoryeffectonHDACexpression
• Thereforenotaffectingthecommensalmicrobiotawhichprotectsandstrengthensoverallskinhealth
• Helpachievethegoalofdevelopingnaturalantimicrobialcosmeticproductswhicharenotharmfulundernormalorforeseeableconditionsofuse
NATURALANTIMICROBIALS
AntimicrobialPeptides
• Exampleofanaturalantimicrobialequippedtokillpathogenicbacteriawhilestillmaintainingavigorouscommensalmicrofloraontheskinareantimicrobialpeptides– Nisin,Natamycin
• Derivedfromfermentationwithprovenefficacy
• Typicallyshortchainsoflessthan50aminoacids
• Synthesizedbyanumberoforganismsasprotectiveorcompetitiveadvantagemechanisms
• Viafermentationtechnology,thesepeptidesaredesigned toprovide:• Superiorantimicrobialefficacy• PromotionofHDACactivityandcommensalbacteriabalance
NATURALANTIMICROBIALS
PeptideFormulaMICResults
PeptideMICresultsdemonstratepotentialeffectivenessatproductadditionratesof1to3%
NATURALANTIMICROBIALS
DoubleChallengeTestResults
ChallengeTestresultsshowsuccessfulprotectionofagenericcreamformulation
PeptideHDACAssayProtocol:HDAC-Glo™AssayandScreeningSystem
• Asingle-reagent-addition,homogeneous,luminescentassaythatmeasurestherelativeactivityofHDACenzymesfromcells
• Usesanacetylated,live-cell-permeant,luminogenicpeptidesubstratethatcanbedeacetylatedbyHDACactivities.
• DeacetylationofthepeptidesubstrateismeasuredusingacoupledenzymaticsysteminwhichaproteaseintheDeveloperReagentcleavesthepeptidefromaminoluciferin
• ThisisquantifiedinareactionusingUltra-Glo™RecombinantLuciferase
• TheHDAC-mediatedluminescentsignalispersistentandproportionaltodeacetlyaseactivity,whichallowsforprocessingofmultiwellplates
ANTIMICROBIALPEPTIDE:HDACDATA
PeptideHDACAssay
Protocol:HDAC-Glo™AssayandScreeningSystem
1. HumanKeratinocytesplatedonDay12. Cellsincubatedongrowthmediaovernight3. Day2– Begin1:2serialdilutions onseparatemultiwellplate4. HDACBufferaddedtoeachwellondilutionplate5. Mediadiscardedfromcellplatetoreplacewithfreshmedia6. Dilutionswithstandardtransferredontocellplate7. Mixforonehourtohomogenize8. Incubatedfor30minutestoallowHDACinhibitortobegin9. Luminescentreagentprepared10. Reagentaddedtocellplate11. Incubatedfor30minutesatroomtemp(23-25C)12. HDACactivitymeasuredoncolorimetricplatereader
ANTIMICROBIALPEPTIDE:HDACDATA
PeptideHDACAssayResults
ANTIMICROBIALPEPTIDE:HDACDATA
PeptideHDACAssayResults
ANTIMICROBIALPEPTIDE:HDACDATA
1.56nM
0
500
1000
1500
2000
2500
3000
Luminescence
HDACAssay
Peptide Phenonip Trichostatin A Triclosan
PeptideHDACAssayResults
ANTIMICROBIALPEPTIDE:HDACDATA
• Demonstratessuccessfulrelationshipbetweenantimicrobialpeptideandskinmicroflora.
• Although theantimicrobialpeptidestillhasslighteffectonHDACactivity,itdoesnotinhibitorreduceHDACnearlyasmuchastriclosanorparabens
• TraditionalpreservativesandbiocidesareshowtodrasticallyinhibitHDACactivity,whichnegativelyaffectsskin’snaturalmicroflorabalance
• Withnaturalantimicrobialpeptide, theskin’scommensalbacterial,whichprotectsagainstunwantedmicrobes,remainseffectiveandintact
TransformingTheFaceOfPreservation:PeptideTechnology
AntimicrobialPeptideSystems
LacticAcidBacteria(LAB)Family
Producenovelantimicrobialpeptides– Bacteriocins
• Proteinaceousmaterials
• Inhibitgrowthofbacterialstrains
• Structurally,functionally,andecologicallydiverse
TransformingTheFaceOfPreservation:PeptideTechnology
Leuconostoc/RadishRootFermentFiltrate
Thenextgenerationofproactive,innovativeandefficacious antimicrobialsystems
TransformingTheFaceOfPreservation:PeptideTechnology
Leuconostoc/RadishRootFermentFiltrate
Mechanism of ActionLAB family – Leuconostoc kimchii produces lactic acid
Restricts the growth ofmicroorganisms by acidifying their environment
Fermentation of Leuconostoc in a defined growth medium containingRadish creates bacteriocins
• Provides broadspectrumantimicrobial activity• Proven conditioningbenefits
ModulatedActivityWillowBark Extract is added – facilitates controlledcell lysis
Ensures the release of the antimicrobialpeptidesformaximised activity
TransformingTheFaceOfPreservation:PeptideTechnology
Leuconostoc/RadishRootFermentFiltrateChallengeTestinaCreamFormulation
MIC %
E.coli 1.60
S.aureus 0.80
P.aeruginosa 1.60
C.albicans 0.80
A.brasiliensis 2.40
MICData
Table1:ChallengeTestResultsfor2.00%Leuconostoc/RadishRootFermentFiltrateinO/WCreamformulationinoculatedonday0andre-inoculatedonday28
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFerment
Mechanism of ActionLAB family – Lactobacillus bulgaricus produces lactic acid
Restricts the growth ofmicroorganisms by acidifying their environment
Fermentationof Lactobacillus creates bacteriocins• Provides broadspectrumantimicrobial activity• Proven conditioningbenefits
ModulatedActivityLysozyme is added to the ferment filtrate – facilitates controlledcell lysis
Ensures the release of the antimicrobialpeptidesformaximised activity
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFermentChallengeTestinaCreamFormulation
MIC %
E.coli 0.75
S.aureus 1.00
P.aeruginosa 1.00
C.albicans 0.75
A.brasiliensis 1.00
MICData
Table2:ChallengeTestResultsfor2.0%LactobacillusFermentinO/WCreamformulationinoculatedonday0andre-inoculatedonday28
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFerment
PCPC Method 3 – Determination of Preservation Adequacy of Water –Miscible Cosmetic and Toiletry FormulationsGenericBase Cream+ 2.0% LactobacillusFerment – pH 3, 4, 5, 6, 7, 8, and 9
PurposeThese studies were initiated to determine the efficacy of a cosmeticingredient with antimicrobial properties in cream formulations againstbioburdenas a function of time.
TestedOrganisms• Escherichia coli• Pseudomonas aeruginosa• Staphylococcus aureus• Aspergillus brasiliensis
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFermentChallengeTestinGenericBaseCream–pH3
Table3:ChallengeTestResultsforGenericCreamwith2.0%LactobacillusFermentinoculatedonDay0.Resultsshowpercent(%)reductioninviableorganisms.
The results of this Challenge Testdemonstrate the effectiveness ofLactobacillus Ferment as an antimicrobialagent in a cream formulation pH 3.
The gram negative and gram positivebacteria were reduced by greater that99.9% within 7 days of each challenge.The mould was reduced by greater than90.0%within 7 daysof each challenge.
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFermentChallengeTestinGenericBaseCream–pH5
The results of this Challenge Testdemonstrate the effectiveness ofLactobacillus Ferment as an antimicrobialagent in a cream formulation pH 5.
The gram negative and gram positivebacteria were reduced by greater that99.9%within 7 days of each challenge. Themould was reduced by greater than 90.0%within 7 daysof each challenge.
Table5:ChallengeTestResultsforGenericCreamwith2.0%LactobacillusFermentinoculatedonDay0.Resultsshowpercent(%)reductioninviableorganisms.
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFerment
The results of this Challenge Testdemonstrate the effectiveness ofLactobacillus Ferment as an antimicrobialagent in a cream formulation pH 7.
The gram negative and gram positivebacteria were reduced by greater that 99.9%within 7 days of each challenge. The mouldwas reduced by greater than 90.0% within 7days of each challenge.
ChallengeTestinGenericBaseCream–pH7
Table7:ChallengeTestResultsforGenericCreamwith2.0%LactobacillusFermentinoculatedonDay0.Resultsshowpercent(%)reductioninviableorganisms.
TransformingTheFaceOfPreservation:PeptideTechnology
LactobacillusFerment
The results of this Challenge Test demonstratethe effectiveness of Lactobacillus Ferment asan antimicrobial agent in a cream formulationpH 9.
The gram negative and gram positive bacteriawere reduced by greater that 99.9% within 7days of each challenge. The mould wasreduced by greater than 90.0% within 7 daysof each challenge.
Table9:ChallengeTestResultsforGenericCreamwith2.0%LactobacillusFermentinoculatedonDay0.Resultsshowpercent(%)reductioninviableorganisms.
ChallengeTestinGenericBaseCream–pH9
NATURALANTIMICROBIALS
• Thehistoryofthecosmeticindustryoverthepast30yearshascreatedincreasingpressureonconventionalpreservativesandbiocides
• Thiswillcontinuetointensifyunlesswefindalternativeapproachestothepreservationofcosmeticsandpersonalcareproducts
• Thereareeffectivenaturalalternativestoconventionalpreservationmethodsthatonlytargetmicrobesbutnottheunderlyingenzymesandcellfunctions theyaffect,suchasHDAC
• Providesasolutiontofindingtheidealbalancebetweeneliminatingbadandpromotinggoodbacteria
Conclusion