viability of dried probiotic strains - eurofoodwater.eu · spray dried log phase l. paracasei nfbc...

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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-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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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

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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

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Storage time (days)

log 10

(cfu

/ml)

Lactobacillus rhamnosus GG Lb338

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Storage time (days)

log 10

(cfu

/ml)

Lactobacillus paracasei 338

Effect of storage RVP on survival of freeze-dried probiotics in skim milk (RSM)

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Storage time (days)lo

g 10(

cfu/

ml)

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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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Storage tim e (days )

log 10

(cfu

/ml)

T re h a lso e syste m

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1 2

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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

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1 2

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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

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1 2

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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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% R

elat

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O.D

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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.

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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