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W. Kneifel © 2010
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
W. Kneifel © 2010
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
W. Kneifel © 2010
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
W. Kneifel © 2010
PRO
DU
CT
PRO
DU
CT
Tech
nolo
gyTe
chno
logy
„„FromFrom strainstrain to to productproduct““
SelectionSelectionStrainStrain
GRAS/QPS
GRAS/QPS
Stabilit
y
Stabilit
y, , Viab
ility
Viabilit
y
Laborat
ory Models
Laborat
ory Models
Dynam
ic
Dynam
icModels
Models
Animal
Animal
Models
Models
Clin
ical
Clin
ical
Stu
dies
Stu
dies
ProbioticProbiotic ProductProduct DevelopmentDevelopment
To To bebe testedtested::•• StrainStrain•• ProductProduct((strainstrain + + matrixmatrix))
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Lag
Phas
eLa
g Ph
ase
Acc
eler
atio
nA
ccel
erat
ion
Phas
ePh
ase
Expo
nent
ial
Pha
seEx
pone
ntia
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P
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Ret
arda
tion
Phas
eR
etar
datio
n Ph
ase
Stat
iona
rySt
atio
nary
Phas
ePh
ase
DieDie
-- offoff P
hase
Phas
e
Lifespan of a Lifespan of a MicrobeMicrobeC
ell
Cel
l Cou
ntC
ount
TimeTime
StabilityStability ViabilityViability
W. Kneifel © 2010
LIVE DEAD
Viab
leVi
able
, , act
ive
activ
e
Viab
leVi
able
. . butbu
t les
sle
ssac
tive
activ
e
Viab
leVi
able
,, butbu
t notno
t im
med
iate
lyim
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iate
lycu
ltura
ble
cultu
rabl
e
Viab
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-- butbu
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t suf
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ntly
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cien
tlycu
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cultu
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e
Viab
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able
, , butbu
tno
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cultu
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Dea
dD
ead
& & n
onnon --
cultu
rabl
ecu
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ble
Cul
tura
ble
Cul
tura
ble
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))
*)*)
*)*)
W. Kneifel © 2010
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
Res
idua
l R
esid
ual c
ount
coun
t(( C
olon
Col
on))
Co n
sum
ptio
nC
o nsu
mpt
ion
„DigestionLoss“
Fres
hFr
esh
prod
uct
prod
uct
Sto r
edSt
o red
pro d
uct
pro d
uct
„StorageLoss“
Res
idua
l R
esid
ual c
ount
coun
t(( C
olon
Col
on))
Co n
sum
ptio
nC
o nsu
mpt
ion
Fres
hFr
esh
prod
uct
prod
uct
Sto r
edSt
o red
pro d
uct
pro d
uct
W. Kneifel © 2010
ViableViable Count Count LossesLosses of of ProbioticProbiotic ProductsProducts
Fres
hFr
esh
prod
uct
prod
uct
Fres
hFr
esh
prod
uct
prod
uct
Res
idua
l R
esid
ual c
ount
coun
t(( C
olon
Col
on))
Co n
sum
ptio
nC
o nsu
mpt
ion
„DigestionLoss“
Res
idua
l R
esid
ual c
ount
coun
t(( C
olon
Col
on))
Co n
sum
ptio
nC
o nsu
mpt
ion
„StorageLoss“
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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
W. Kneifel © 2010
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
W. Kneifel © 2010
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
W. Kneifel © 2010
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
W. Kneifel © 2010
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
W. Kneifel © 2010
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??
W. Kneifel © 2010
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
W. Kneifel © 2010
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
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