Preservation of probiotic bacteria by freeze-drying, and achieving stomach and bile acid resistance

Dr Kevin R. WardDirector of Research & Development, BTLWinnall Valley Road, Winchester SO23 0LD

T: 01962 841092E: kward@biopharma.co.ukW: www.btl-solutions.net

Synopsis

• What is freeze-drying?

• Freeze-drying as a method of preservation

• Current issues with probiotic products• Current issues with probiotic products

• Objectives of our TSB-sponsored project

• Results and Achievements of the project

• Next steps

What is Freeze-Drying?

• Freeze-drying (lyophilisation) is the removal of frozen solvent by sublimation under vacuum, and unfrozen solvent by desorption

• Materials should be characterised and • Materials should be characterised and freeze-dried below their intrinsic critical temperature (Tc / Teu)

• A wide range of materials across the pharmaceutical, diagnostics and food sectors are freeze-dried

Freeze-drying as amethod of preservation

• Freeze-drying was first used commercially in the 1940s (plasma, antibiotics). It is now well established as a means of preservation for:– Small molecules (e.g. traditional drug molecules)– Small molecules (e.g. traditional drug molecules)– Proteins (including enzymes, antibodies)– Foods (e.g. coffee, milk, backpackers’ meals)– Plasma components, hormones– Whole cells, organisms, viruses, bacteria (but not

probiotic bacteria, although some starter cultures in the dairy industry are freeze-dried)

Current issues with “Probiotic Yogurt Drinks”• Probiotic yogurt drinks require refrigeration

during transport and storage

• Manufacturers only guarantee number of live organisms at point of manufacturelive organisms at point of manufacture

• Organisms still viable at point of use need to survive stomach acid and bile acids

• Therefore, a product containing a defined number of bacteria that could be delivered to the gut would be an advantage

Objectives of TSB Project

• To produce stable L. crispatus (a challenging organism!) efficiently that can be delivered successfully to the gut:

• Fermentation: ‘optimisation’ of conditions to give >20% increase in harvest compared to current productionharvest compared to current production

• Formulation & Freeze-Drying: to give less than 1 log reduction in viability and good storage stability at ambient temperature

• Gut Delivery Systems: investigating natural and synthetic bile acid resistant (BAR) reagents for co-encapsulation with bacteria

FermentationVariables Studied

• Culture medium components– Modification by addition of a range of adjuncts

• Physical parameters• Physical parameters– Temperature– pH

• Point of harvest comparisons– Early log phase– Late log phase– End log phase– Stationary phase

FermentationResults

• The best combination of harvest time, media constituents and physical parameters gave >20% improvement in parameters gave >20% improvement in viable counts over historical batches

• Scale up to 1200L fermentation batch using these conditions gave a further improvement in harvestable yield

Formulation & Freeze-DryingVariables Studied

• Bulking / protective agents:– Twelve candidates examined– Compared with absence of protective agent– Method of addition (dissolving into slurry vs.

mixing slurry with concentrated solutions)

• Freeze-Drying conditions:– Cycles based on collapse temperature of

each formulation, with a view to making cycles as efficient as possible

Formulation & Freeze-DryingResults from 5 runs

Formulation: 1 2 3 4 5 6 7 8 9 10 11 12 C1 C2 S

Bile Acid Resistant reagentsVariables Studied

• Eight petrochemical and naturally occurring BARs (from renewable sources) were studied and compared against positive & negative controls

• BARs were subjected to a simulated bile solution for 60 mins to assess extent & rate of degradation

• The 3 best-performing BARs were then combined with freeze-dried powders and dosed into enteric coated capsules for degradation / stability studies

BAR reagentsResults

• Using a combination of different BAR reagents yielded protection of L. crispatusagainst bile acid:– >1000 x better protection than negative control– >1000 x better protection than negative control– >100x better protection than positive control

• The best BAR combination comprised:– 40% Natural Product Component 1– 40% Natural Product Component 2– 20% Filler

Summary of ResultsAt end of First 3 months

• Fermentation Studies: >20% increase in harvestable yield achieved

• Formulation & Freeze-Drying Studies: Less than 1 log reduction in viability achieved, using a standard food excipient, 6 day cyclestandard food excipient, 6 day cycle

• BAR Reagent Studies: Some BAR reagents showed promise in protecting L. crispatus against acids for 60 mins, allowing rehydration and revival

The above knowledge was then combined in the final3 months of the project to produce an ‘optimised pr oduct’

‘Optimised Product’Results (1)

• Process scaled up:– 1200L fermentation– Formulation– Freeze-Drying– Encapsulated with BAR– Encapsulated with BAR

• Early stability data:– Promising for freeze-dried

bacteria– <1 log drop observed after

14d at 37°C– >50% viability observed

after 14d at 25°C

‘Optimised Product’Results (2)

• Stability of the material after grinding and encapsulation with BAR reagent was not as promising as the dried material itself

• Effect of grinding was investigated but material seemed stable to this process

• Subsequent data suggests transfer of moisture from BAR to bacteria within capsule

• This issue warrants further investigation

Next Steps

• Apply the same development process to other bacteria requiring stabilisation and/or delivery to the gut in a viable state using an oral capsule dosage formoral capsule dosage form

• Take the current process to the next level using microencapsulation to produce a similarly stable product in liquid suspension dosage form or for incorporation into foods

Acknowledgements

Fermentation studies:Tim NelsonDr David Reynolds

Formulation andFreeze-Drying studies:Isobel CookMervyn Middleton

Sponsorship:

BAR reagent studies:Prof. Nigel SlaterKrishnaa Mahbubani

Mervyn MiddletonTom Peacock

Presented by Dr Kevin Ward, BTL

www.btl-solutions.netbtl@biopharma.co.uk