viability of dried probiotic strains - eurofoodwater.eu · spray dried log phase l. paracasei nfbc...
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Life without WaterViability of dried probiotic strains
Life without WaterViability of dried probiotic strains
SummaryProbiotics: Myth or RealityThe Need for Dry Viable CulturesFreeze Drying and Spray DryingViability and W ater ActivityViability and Cell PhysiologyOverall Conclusions
Definition“live microorganisms which when
consumed in adequate numbers confer a health benefit on the host”
FAO/WHO Expert Consultation 2001
ProbioticsBifidobacterium•anaerobic•complex nutritional requirements•unique carbohydrate metabolism (F-6-P shunt)•utilize complex polysacs (prebiotics)
Lactobacillus•facultative•complex nutritional requirements•homo/hetero lactic fermentation•easier to cultivate•more acid resistant
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9
10
-2 -1 1 2 3 4 5 6 7 8 9 10 11 12 weeks
placebo
Lactobacillus
BifidobacteriumComposite symptom score
Treatment period
Preliminary Probiotic IBS Treatment Study
(Double-blind placebo controlled design)
*
** * * *
** P<0.05
Irritablebowel
syndrome
O’Mahony et al, Gastroenterology, 2005
Life without WaterViability of dried probiotic strains
SummaryProbiotics: Myth or RealityThe Need for Dry Viable CulturesFreeze Drying and Spray DryingViability and W ater ActivityViability and Cell PhysiologyOverall Conclusions
Viability is everything!
Paul Ross1Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co.
Cork2Alimentary Pharmabiotic Centre, Cork
3Department of Microbiology, University College, Cork, Ireland
Hamilton-Miller et al. , 1999
•The Problem:Generating high viability (1 0 9 /g) powders
Application of Lactobacillus spray dried powder in Cheddar cheese manufacture
Starter lactococci
Lb. paracasei NFBC 338 Rifr
NSLAB (Control cheese)
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9
10
0 30 60 90Ripening time (days)
Log
CFU
/g
4 X 1 0 6 CFU/ml
1 .1 x 1 0 9 CFU/g
Gardiner et al. 2004 Lett Appl. Microbiol
Life without WaterViability of dried probiotic strains
SummaryProbiotics: Myth or RealityThe Need for Dry Viable CulturesFreeze Drying and Spray DryingViability and W ater ActivityViability and Cell PhysiologyOverall Conclusions
Industrial Generation of Probiotics: freeze-drying
Freeze drying
Generation of Probiotics: spray-drying
Spray drying
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
7 0 -7 5 7 5 -8 0 8 0 -8 5 8 5 -9 0 9 0 -9 5 9 5 -1 0 0 1 0 0 -1 0 5 1 2 0
Outlet temperature (oC)
0123456789
% su
rviv
al
% m
oist
ureLb. paracasei NFBC 338
85oC 100oC
Spray-Drying Conditions
Gardiner et al, Appl. Environ Microbiol
Freeze dried log phase L. paracasei NFBC 338, control(x60,000).
Spray dried log phase L. paracasei NFBC 338, overproducing GroESL (x60,000)
Spray dried log phase L. paracasei NFBC 338, control(x60,000).
Freeze dried log phase L. paracasei NFBC 338, control(x60,000).
Life without WaterViability of dried probiotic strains
SummaryProbiotics: Myth or RealityThe Need for Dry Viable CulturesFreeze Drying and Spray DryingViability and W ater ActivityViability and Cell PhysiologyOverall Conclusions
Effect of storage RVP on survival of freeze-dried probiotics
0%RVP (P2O5)
11.4%RVP (LiCl)
33.2%RVP (MgCl2)
44.1%RVP (K2CO3)
76.1%RVP (NaCl)
Lactobacillus paracaseissp. paracasei NFBC338
LGG
0
2
4
6
8
10
0 5 10 15 20 25 30 35 40
Storage time (days)
log 10
(cfu
/ml)
Lactobacillus rhamnosus GG Lb338
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6
8
10
12
0 5 10 15 20 25 30 35 40
Storage time (days)
log 10
(cfu
/ml)
Lactobacillus paracasei 338
Effect of storage RVP on survival of freeze-dried probiotics in skim milk (RSM)
0
2
4
6
8
10
0 5 10 15 20 25 30 35 40
Storage time (days)lo
g 10(
cfu/
ml)
0
2
4
6
8
10
0 5 10 15 20 25 30 35 40
Storage time (days)
log 10
(cfu
/ml)
Lactobacillus rhamnosus GG Lactobacillus paracasei 338
0%RVP (P2O5)11.4%RVP (LiCl)
33.2%RVP (MgCl2)
44.1%RVP (K2CO3)
76.1%RVP (NaCl)
Freeze-drying Lactobacillus rhamnosus GG in the presence of disaccharides
Control Lactose Trehalose Maltose Sucrose L/T mix L/M mix
Mean Log10(cfu/ml) ± SD
AfterFreezedrying
8.23±0.10 *
8.92±0.02 **
9.12±0.12 **
9.02±0.25 **
8.65±0.21*
9.12±0.31 **
9.20±1.10 **
Beforefreezin
9.36±0.12 9.52±0.7 9.39±0.89 9.36±0.71 9.78±0.31 9.35±0.52 9.32±1.21
Afterfreezin
9.01±0.02*
9.03±0.08*
9.37±1.01 **
9.00±0.02 *
9.55±0.71 **
9.35±0.12 **
9.23±0.32 **
* Significant and ** no significant (P≤0.05)
Effect of storage RVP on survival of freeze-dried probioticbacteria in presence of Mix Sugar
S ucrose syste m
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8
10
12
0 5 10 15 20 25 30 35 40 45 50
Storage tim e (days )
log 10
(cfu
/ml)
T re h a lso e syste m
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5
S to r a g e t im e ( d a y s )
og10
(cu/
)
Trehalose Sucrose
L a c to se /M a l to se syste m
0
2
4
6
8
1 0
1 2
0 1 0 2 0 3 0 4 0 5 0 6 0
S t o r a g e t im e (d a y s )
log 10
(cfu
/ml)
Lactose/maltose
More cell death1 1 .4 %
3 3 .2 %
RVP Values
0 %
11.4%
3 3 .2 %
4 4 .1 %
7 6 .1 %L a cto se /T re h a lso e syste m
0
2
4
6
8
1 0
1 2
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0
S to r a g e t im e ( d a y s )
g 10(
)
Lactose/Trehalose
Glass transition temperatures of the model systems at various RVP
RVP RSM Lactose Trehalose Maltose Sucrose L/T mix L/M mix
0% 98.1±1.0 106.2±0.4 107.9±1.1 90.6±0.7 51.2±0.3 107.3±0.5 96.9±1.2
11% 58.2±0.5 55.7±1.0 53.8±1.0 46.0±1.3 31.6±0.7 53.6±1.2 51.3±0.3
23% 34.2±1.3 40.4±0.2 40.4±0.1 31.7±0.9 21.6±0.2 40.1±0.3 33.0±1.1
33% 20.1±0.3 30.4±0.4 29.5±0.9 23.0±0.1 C 30.1±0.2 25.9±0.4
44% C C C 8.2±0.4 C 11.8±0.2 10.0±0.6
*C- system crystallized
Below Room Temp
Above Room Temp
Effect of glass transition temperature on the cell activity
1 .0 0 E+0 0
1 .0 0 E+0 2
1 .0 0 E+0 4
1 .0 0 E+0 6
1 .0 0 E+0 8
1 .0 0 E+1 0
-1 0 0 -8 0 -6 0 -4 0 -2 0 0
T-Tg
Lactose/Trehalose
Cell Viability (Colony Forming Units/ml)
Life without WaterViability of dried probiotic strains
SummaryProbiotics: Myth or RealityThe Need for Dry Viable CulturesFreeze Drying and Spray DryingViability and W ater ActivityViability and Cell PhysiologyOverall Conclusions
The strain itself: Technological Criteria
• Culturable to high cell density• Grow in dairy-based media• Stress tolerant (Oxygen Acid Salt and Heat)
• Robust to concentration and preservation• No off-flavours etc.• Compatible with product starter/flora • Maintain probiotic properties• Phage resistance
0102030405060708090
100
% R
elat
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Surv
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Not all bifidobacteria behave the same
Heat tolerance of different bifidobacteria species (57oC X 5 min)
0102030405060708090
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Oxygen tolerance of bifidobacteria
Gardnerella vaginalis
B. magnum
B. subtileB. breve
B. bifidum
B. pullorum
B. asterodies
B. pseudolongumsubsp . globosum
B. choerinum
B. thermophilum
B. infantis
B. dentium
B. pyschraerophilum
B. pseudolongumsubsp . pseudolongum
B. animalissubsp . lactisB. animalissubsp . animalisB. cuniculi
B. gallicumB. saeculareB. gallinarum
B. longumB. suis
B. thermacidophilmB. boum
B. adolescentisB. ruminantium
B. catenulatumB. pseudocatenulatum
B. angulatumB. merycicum
B. minimum
B. indicumB. coryneforme
Scardovia inopinataParascardovia denticolens
Aeriscardovia aeriphila
Gardnerella vaginalis
B. magnum
B. subtileB. breve
B. bifidum
B. pullorum
B. asterodies
B. pseudolongumsubsp . globosum
B. choerinum
B. thermophilum
B. infantis
B. dentium
B. pyschraerophilum
B. pseudolongumsubsp . pseudolongum
B. animalissubsp . lactisB. animalissubsp . animalisB. cuniculi
B. gallicumB. saeculareB. gallinarum
B. longumB. suis
B. thermacidophilmB. boum
B. adolescentisB. ruminantium
B. catenulatumB. pseudocatenulatumB. angulatum
B. merycicumB. minimum
B. indicumB. coryneforme
Scardovia inopinataParascardovia denticolens
Aeriscardovia aeriphila
Gardnerella vaginalis
B. magnum
B. subtileB. breve
B. bifidum
B. pullorum
B. asterodies
B. pseudolongumsubsp . globosum
B. choerinum
B. thermophilum
B. infantis
B. dentium
B. pyschraerophilum
B. pseudolongumsubsp . pseudolongum
B. animalissubsp . lactisB. animalissubsp . animalisB. cuniculi
B. gallicumB. saeculareB. gallinarum
B. longumB. suis
B. thermacidophilmB. boum
B. adolescentisB. ruminantium
B. catenulatumB. pseudocatenulatumB. angulatum
B. merycicumB. minimum
B. indicumB. coryneforme
Scardovia inopinataParascardovia denticolens
Aeriscardovia aeriphila
HEAT OXYGEN SPRAY DRYING
HEAT (57oC)Survival > 1%
Survival < 1% Species not examined
Growth < 5%Growth > 5%
Species not examined
OxygenStress resistance related to their genetic make-up
Simpson et al, 2005, J. Appl. Microbiol.
Pre-adaption improves survival during spray drying
-505
1 01 52 02 53 0
9 5 - 1 0 0 1 0 0 - 1 0 5
Outlet Temperature
% S
urvi
val
Control
Heat-Adapted
-1 0
0
1 0
2 0
3 0
4 0
9 5 - 1 0 0 1 0 0 - 1 0 5
Outlet Temperature
% S
urvi
val
Control
Salt-AdaptedSalt(0.3 M NaCl X 30min)
Heat(52oC X 15min)
4.8 x 107 8.02 x 107 1.8 x 1062.4 x 107
2.44 x 108 6.99 x 108 2.02 x 108 5.95 x 109
Desmond et al. 2001 Appl. Environ. Micrbiol.
37oC
GroEL
52oC X 15min
Model for a GroEL-GroES folding reaction
pGro2pC002
Time + nisin 0 1 3 5 0 1 3 5
pGro1pC001
Time + nisin 0 1 3 5 0 1 3 5
GGAGGGATTGCCSph1 Xba1
RBSgroES groELPnis
A B
pNZ8048 (3394)
GroEL66Kda
GroEL66Kda
Overexpressed GroESL in Cheese Starter and Probiotic
Fig. 3 SDS PAGE illustrating the controlled expression of groESL using the NICE system in (A) L.lactis NZ9800 using pNZ8048, and (B) Lb. paracasei NFBC 338 using pMSP3535. Desmond et al.,2004, Appl. Environ. Microbiol.
0
1
2
3
4
5
6
Control groESL
Perc
ent s
urvi
val
Survival of Lb. paracasei subsp. paracasei NFBC 338 following spray drying (T0 = 95-100oC, n=3)
GroESL-overproducing strain
Vector control
Corocran et al.,2005, Appl. Environ. Microbiol.
Life without WaterViability of dried probiotic strains
Conclusions
Need for Dry Viable Cultures Stable at Room Temp
Some Strains Survive Spray Drying
Increased Relative Humidity = Decreased Viability
High Glass Transition Temp = Stable Probiotics
Increase Cellular Stress Protection = Increased Viability
Thanks!Viability of dried probiotic strains
Dr. Song MiaoDr. Collette DesmondDr. Barry CorcoranDr. Paul Simpson
Dr. Gillian Gardiner
CollaboratorsDr Catherine Stanton
Prof. Yrjö H. RoosProf. Ger Fitzgerald