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RoleRole of Food Matrixof Food Matrixforfor ProbioticProbiotic EffectsEffects

W. W. KneifelKneifel

Department of Food Science and TechnologyDepartment of Food Science and Technology

BOKU BOKU –– University of University of NaturalNatural Resources and Life Sciences Resources and Life Sciences ViennaVienna

Hohenheim, 15 Hohenheim, 15 OctOct. 2010. 2010

wolfgang.kneifel@boku.ac.atwolfgang.kneifel@boku.ac.at

W. Kneifel © 2010New New NutritionNutrition Business Vol. 15, no.9, Business Vol. 15, no.9, JulyJuly 20102010

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TheThe SpectrumSpectrum of of ProbioticProbiotic ProductsProducts

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MAIN MESSAGESMAIN MESSAGESrelatedrelated toto

DifferingDiffering Formats and Formats and PropertiesPropertiesof of ProbioticsProbiotics

ViabilityViability and and StabilityStability as Key as Key IssuesIssuesforfor ApplicationsApplications –– ComparativeComparative EvaluationEvaluation

TailoringTailoring ProbioticsProbiotics -- IndividualIndividual RequirementsRequirementsand and SynergiesSynergies, , whatwhat LiteratureLiterature TellsTells

ConcludingConcluding RemarksRemarks and and ChallengesChallenges

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•• …… possessingpossessing scientificscientific evidenceevidence regardingregardingbeneficialbeneficial effectseffects exertedexerted to to thethe individualindividual

•• …… possessingpossessing accurateaccurate statestate--ofof--thethe--artarttaxonomictaxonomic characterisationcharacterisation

•• …… possessingpossessing evidenceevidence to to reachreach thethe targettarget regionregionat at high high cellcell densitydensity, , viabilityviability and and stabilitystability

•• …… fulfillingfulfilling all all safetysafety requirementsrequirements accordingaccording to to scientificscientific statestate--ofof--thethe--artart ((GRASGRAS,, QPSQPS))

ProbioticsProbiotics areare wellwell--selectedselected

as as onlyonly thosethose strainsstrains……

fulfilfulfil thethe qualityquality criteriacriteria FDAFDA

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FunctionalityFunctionality

IdentityIdentity

SafetySafety

StabilityStability

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DEFINITIONDEFINITION OF PROBIOTICS:OF PROBIOTICS:

….... are live microorganisms which whenadministered in adequate amounts confera health benefit to the host…….

analyticalanalytical and and technologicaltechnological challengeschallengesforfor productproduct designdesign and and developmentdevelopment!!

FunctionalityFunctionality and and StabilityStability

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PRO

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PRO

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„„FromFrom strainstrain to to productproduct““

SelectionSelectionStrainStrain

GRAS/QPS

GRAS/QPS

Stabilit

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Viabilit

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Laborat

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ProbioticProbiotic ProductProduct DevelopmentDevelopment

To To bebe testedtested::•• StrainStrain•• ProductProduct((strainstrain + + matrixmatrix))

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Lifespan of a Lifespan of a MicrobeMicrobeC

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TimeTime

StabilityStability ViabilityViability

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LIVE DEAD

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TechnoTechno--analyticalanalytical approachapproach

CFUsCFUs

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10 10 -- 10 CFU/g10 CFU/g1010 1111

10 10 -- 10 CFU/g10 CFU/g66 99

LyophilizedLyophilized bacteriabacteriaStabilizedStabilized, (, (cryocryo--))protectantsprotectantsEncapsulatedEncapsulated ((resistantresistantagainstagainst gastricgastric juicejuice))

ActiveActive bacteriabacteriain in soursour proteinprotein gelgel matrixmatrixAdditional Additional protectantsprotectants

! ! ConsumptionConsumption portionportion !!! ! ConsumptionConsumption frequencyfrequency !!

! ! InteractionsInteractions !!

MicrobialMicrobial CellCell DensityDensity –– ViableViable countscounts

*) *) viableviable countcount((plateplate countcount methodmethod,,

standardizedstandardized))

*)*)

*)*)

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FluorescentFluorescent stainingstaining of of L. L. caseicasei((Live/DeadLive/Dead statusstatus))

XX--GluGlu agaragar plateplatewithwith coloniescolonies of of

L.acidophilusL.acidophilus ((blueblue) and) andL. L. caseicasei ((whitewhite))

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ViableViable Count Count LossesLosses of of ProbioticProbiotic ProductsProducts

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ViableViable Count Count LossesLosses of of ProbioticProbiotic ProductsProducts

Fres

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InherentInherent productproductcompositioncomposition

TechnologyTechnology

Milieu Milieu conditionsconditions

IndividualIndividual strainstraincharacteristicscharacteristics

SelectedSelected cultureculture typetype

Special Special ingredientsingredients

StabilityStability and and resistanceresistance factorsfactors,,„„StressStress--trainingtraining experienceexperience““,,

cellcell--wallwall compositioncomposition

DeepDeep--frozenfrozen concentratedconcentrated cultureculture,,lyophilizedlyophilized cultureculture,,

(liquid (liquid cultureculture))

ProteinProtein--fatfat matricesmatrices, , sugarsugar concentrationconcentration,,

hydrocolloidhydrocolloid agentsagents etc.etc.

PrebioticPrebiotic carbohydratescarbohydrates((……synbioticssynbiotics), ), fruitfruitpreparationspreparations, , otherother

SeparatedSeparated fermentationfermentation,,coco--fermentationfermentation, ,

postpost--fermentationfermentation supplementationsupplementation

RedoxRedox statusstatus ((oxygenoxygen),),temperaturetemperature, , pHpH, , acidityacidity, time, time

DAIRYDAIRYPRODUCTSPRODUCTS

TALKING ABOUTTALKING ABOUT

W. Kneifel © 2010

PHARM.PHARM.PRODUCTSPRODUCTS

InherentInherent productproductcompositioncomposition

TechnologyTechnology

Milieu Milieu conditionsconditions

IndividualIndividual strainstraincharacteristicscharacteristics

Special Special ingredientsingredients

StabilityStability and and resistanceresistance factorsfactors,,„„StressStress--trainingtraining experienceexperience““,,

resuscitationresuscitation propertiesproperties,,cellularcellular compositioncomposition

ProteinProtein--carbohydratecarbohydrate matricesmatrices, , cryoprotectantscryoprotectants, , waterwater--bindingbinding

agentsagents

Growth Growth activatorsactivators uponuponrehydrationrehydration

CultureCulture biotechnologybiotechnology,,different different standardsstandards

Water Water activityactivity, , redoxredox statusstatus ((oxygenoxygen),),temperaturetemperature, , pHpH, , acidityacidity, time, time

TALKING ABOUTTALKING ABOUT

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BacteriaBacteria presentpresent in different in different formsforms of of viabilityviability, , mainlymainlyactiveactive, , butbut limitedlimited ((shelfshelf--)life)life

BacteriaBacteria presentpresent in in dormantdormantand and drydry statusstatus, , needneed forforresuscitationresuscitation uponupon applicationapplication

ProteinaceousProteinaceous matrixmatrix maymayexertexert protectionprotection to to thethe bacteriabacteriain in productproduct and in GI and in GI tracttract

Alternative Alternative foodfood carrierscarriers ((otherotherthanthan dairydairy) ) maymay bebe envisagedenvisaged

Different Different techniquestechniques maymay bebeappliedapplied to to preservepreserve bacteriabacteriain in thethe productsproducts on on longlong--termterm((e.ge.g., ., [[micromicro]]encapsulationencapsulation))

Delivery of Delivery of bacteriabacteria stronglystronglydependsdepends on on degreedegree of reof re--hydrationhydration and and rere--activationactivation

ProductProduct acidityacidity supportssupportsresorptionresorption of of nutritivenutritivecompoundscompounds and and mineralsminerals

VS.VS.

Key Key CriteriaCriteria of Different of Different ProbioticProbiotic FormatsFormats

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Key Key QuestionsQuestions ConcerningConcerningDifferent Different ProbioticProbiotic ProductProduct FormatsFormats

Are Are probioticprobiotic effectseffects of of defineddefined strainsstrains different, different, whenwhensamesame strainsstrains areare appliedapplied as as drieddried preparationspreparations oror throughthrougha a fermentedfermented dairydairy productproduct??

CanCan probioticprobiotic effectseffects of of defineddefined strainsstrains bebe enhancedenhanced ififcertaincertain additivesadditives oror ingredientsingredients areare addedadded to to thethe productproduct((oror preparationpreparation)?)?

LITERATURELITERATURE

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Not all Not all lacticlactic bacteriabacteria areare probioticsprobiotics, , ……. . butbut somesome areareLetter to Letter to thethe EditorEditor

AzaisAzais--BraescoBraesco V et al., Brit. J. V et al., Brit. J. NutrNutr. 103 (2010) 1079. 103 (2010) 1079--10811081

Food Food matricesmatrices havehave an an impactimpacton on thethe expressionexpression of of

genesgenes involvedinvolved in in probioticprobiotic--linkedlinked mechanismsmechanisms

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InfluenceInfluence of of carbohydratescarbohydrates on on cellcell propertiesproperties ofofLactobacillusLactobacillus rhamnosusrhamnosus

BegovicBegovic, J et al., , J et al., CentrCentr. . EurEur. J. Biol. 5 (2010) 103. J. Biol. 5 (2010) 103--110110

TheThe presencepresence of of fructosefructose, , mannosemannose,,oror rhamnoserhamnose significantlysignificantly in in thethe

growth growth mediummedium significantlysignificantly reducesreducescellcell surfacesurface hydrophobicityhydrophobicity, ,

mucoiditymucoidity and and cellcell wall wall compositioncompositionof a of a L. L. rhamnosusrhamnosus strainstrain..

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A A combinationcombination of of galactogalacto--oligosaccharidesoligosaccharides and and LactobacillusLactobacillus GG GG increasesincreases bifidobacteriabifidobacteria to a to a greatergreaterextentextent thanthan LactobacillusLactobacillus GG on ist GG on ist ownownKekkonen, RA et al., Milchwissenschaft 62 (2007) 326Kekkonen, RA et al., Milchwissenschaft 62 (2007) 326--330330

DoubleDouble--blind blind crosscross--overover studystudywithwith 44 44 adultsadults, , fruitfruit--milkmilk drinkdrink

withwith LGG LGG withoutwithout and and withwith GOS:GOS:fecalfecal bifidobacterialbifidobacterial countscounts moremore

increasedincreased in LGG+GOS in LGG+GOS groupgroup

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ReconstitutionReconstitution conditionsconditions forfor drieddried probioticprobiotic powderspowdersrepresentrepresent a a criticalcritical stepstep in in determiningdetermining cellcell viabilityviability

Muller JA et al., J. Muller JA et al., J. ApplAppl. . MicrobiolMicrobiol. 108 (2010) 1369. 108 (2010) 1369--13791379

ReconstitutionReconstitution and and cellcell integrityintegrity studystudywithwith BifidobacteriumBifidobacterium longumlongum and and

L. L. johnsoniijohnsonii sstrainstrains::reconstitutionreconstitution conditionsconditions (media, time,(media, time,

protectantsprotectants etc.) etc.) stronglystrongly affectaffect viabilityviability,,activityactivity, , morphologymorphology and and metabolicmetabolic profileprofile

of of probioticprobiotic strainsstrains

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ChallengesChallenges in in thethe additionaddition of of probioticprobiotic culturescultures totofoodsfoods

ChampagneChampagne CP & Gardner J, CP & Gardner J, CritCrit. Rev. Food . Rev. Food SciSci. . NutrNutr. 45 (2005),. 45 (2005),6161--8484

•• TooToo muchmuch sugarsugar and and dissolveddissolved oxygenoxygen in in thethe productproduct resultresultin in reducedreduced probioticprobiotic countscounts and and stabilitystability

•• AscorbicAscorbic acidacid additionaddition mymy resultresult in in increasedincreased L. L. acidophilusacidophiluscountscounts

•• S. S. thermophilusthermophilus in in fermentationfermentation cultureculture possessespossessesoxygenoxygen--scavengingscavenging propertiesproperties

•• L. L. delbrdelbr.. subspsubsp. . bulgaricusbulgaricus in in fermentationfermentation cultureculture maymayreducereduce probioticprobiotic countcount

•• Addition of Addition of proteinprotein hydrolysateshydrolysates and UF and UF retentatesretentates maymaystimulatestimulate growth of growth of certaincertain probioticsprobiotics

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To To thethe consumerconsumer, , productproduct and brandand brand mustmust bothboth bebea a crediblecredible carriercarrier of of thethe benefitbenefit

ThereThere mustmust bebe a a logiclogic to to thethe wholewhole thatthat thethe consumerconsumercancan acceptaccept

ProductProduct mustmust meetmeet sensorysensory preferencespreferences

ProductProduct mustmust fulfilfulfil fundamental fundamental nutritionalnutritional requirementsrequirements

ProbioticProbiotic dairydairy productsproducts ––importantimportant prerequisitesprerequisites

CONSUMERCONSUMER PERCEPTION AND PREFERENCESPERCEPTION AND PREFERENCES

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ProbioticProbiotic PizzaPizza

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ScreeningScreening: : CompositionalCompositional detailsdetailsof of probioticprobiotic drinksdrinks and and yoghurtsyoghurts((AustrianAustrian marketmarket))

n = 26n = 26

CarbohydrateCarbohydrate rangerange::

Protein Protein rangerange::

FatFat rangerange::

4,0 4,0 -- 14,714,7 %%

1,3 1,3 -- 5,2 %5,2 %

0,1 0,1 –– 3,6 %3,6 %

Energy Energy rangerange:: 121 121 –– 413 413 kJkJ

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„…„…perceivedperceived carriercarrier--ingredientingredient fit was fit was relatedrelated to to familiarityfamiliarity withwiththethe combinationcombination and and healthinesshealthiness of of carriercarrier foodfood ((statusstatus DenmarkDenmark))““

Are Are consumersconsumers sufficientlysufficiently informedinformed and and howhow isis theirtheir responseresponse to to functionalfunctional foodsfoods??

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makemake probioticsprobiotics becomebecome visiblevisible to to thethe consumerconsumerChampagneChampagne CP & CP & FustierFustier P: P: CurrCurr. . OpinOpin. . BiotechnolBiotechnol. 18 (2007). 18 (2007)184184--190190

MicroencapsulatedMicroencapsulatedprobioticprobiotic bacteriabacteria

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ConclusionsConclusions

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As As longlong as as itit isis notnot sufficientlysufficiently specifiedspecified forfor a a defineddefinedprobioticprobiotic productproduct and and itsits correspondingcorresponding targettargetapplicationapplication howhow manymany ((viableviable) ) cellscells needneed to to bebe administeredadministered,,itit remainsremains difficultdifficult to to exactlyexactly designdesign a a tailoredtailored productproduct

as as manymany bacteriabacteria as as possiblepossible? ? moremore dosedose--responseresponse studiesstudies,,

DegreeDegree of of matrixmatrix--basedbased effectseffects on on stabilitystability and and viabilityviability of of probioticprobiotic strainsstrains dependsdepends on on individualindividual strainstrain characteristicscharacteristics

moremore comparativecomparative studiesstudies withwith strainsstrains underunder varyingvaryingmatrixmatrix conditionsconditions neededneeded to to optimizeoptimize a a productproduct

FermentedFermented dairydairy productsproducts as as probioticprobiotic carrierscarriers seemseem to to offerofferadditional additional beneficialbeneficial effectseffects to to thethe consumerconsumer, , ifif thethe basicbasicnutrientnutrient profileprofile isis accordingaccording to to statestate--ofof--thethe artart

needneed forfor rere--consideringconsidering original original productproduct formulaeformulae to to improveimprove thethe basicbasic qualityquality of of thethe carriercarrier

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moremore studiesstudies withwith final final productproduct neededneeded