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

Figure 1. Vials of freeze-dried L. crispatus.
Figure 2. Results from five runs showing the 12 candidates examined.
Figure 3. Example of encapsulated end product.

Following the receipt of a grant from the UK Technology Strategy Board (TSB), the British-based company Biopharma Technology Limited (BTL) worked in conjunction with the University of Cambridge and biotechnology specialists Microbial Developments Ltd on a study to investigate methods of improving the delivery of probiotic bacteria.
The project focused on three key areas: optimisation of fermentation methods; freeze-drying formulation and procedure; and a gut delivery system. The aim was to produce stable Lactobacillus crispatus in a format that can be reliably and efficiently delivered to the gut.
At the end of the six-month study, fermentation studies carried out by Microbial Developments Ltd had shown that it was possible to have a 20% increase in the harvestable yield, BTL had designed a formulation and drying cycle that enabled the bacteria to be freeze-dried with a less than 1 log reduction in viability and Bile Adsorbing Resin (BAR) reagents developed by the University of Cambridge showed promise in protecting the bacteria for up to sixty minutes, allowing rehydration and revival.
The results from these studies were combined to produce a complete ‘optimised product’ in capsule form, which will be subject to further investigation in 2011.

by Sarah Webster

The collaboration
The project brought together industrial and academic partners in Microbial Developments Ltd, the University of Cambridge and BTL. The University, led by Professor Nigel Slater, brought specific technology and expertise for the effective formulation of orally-available microbial preparations, particularly in the field of bile acid stabilisers. Microbial Developments is an innovative biotechnology company with 25 years experience in the manufacture, fermentation and freeze-drying of micro-organisms; the company currently exports to 47 countries worldwide. BTL was established in 1997 to provide contract freeze-drying research and development services, training and advanced instrumentation to the global biopharmaceutical and related industries. Since then, BTL has worked with over 700 different materials and the current team has decades of combined experience of in a wide range of applications of freeze-drying technology.

The Technology Strategy Board
The project was co-funded by the TSB through the ‘Developing High Value Chemicals Competition’ launched in 2010. The TSB is a business-led executive non-departmental public body, established by the UK government. Its role is to promote and support research into, and development and exploitation of, technology and innovation for the benefit of UK business, in order to increase economic growth and improve quality of life. It is sponsored by the UK Department for Business, Innovation and Skills (BIS). The Competition was set up to investigate the vailidity of funding projects investigating how industrial biotechnology could be competitively applied to the production of high-value chemicals.
The probiotics problem
‘Friendly’ bacteria are vital to many systems within the human body including the digestive and immune systems. Probiotic supplements have become increasingly popular and probiotic yogurts and yogurt drinks are common in many households. However as a delivery system for probiotics, yogurts have some drawbacks. The number of live organisms that yogurts contain can only be guaranteed at the point of manufacture. Temperature sensitivity and a short shelf life necessitate costly refrigeration during both transport and storage. Finally, any organisms still viable at the point-of-use need to survive the digestive process in order to colonise the gut.

Probiotics are amongst the highest value ingredients in the food industry, yet this poor stabilisation and formulation frustrates supply chains. The stabilised probiotic formulations developed through this project are innovative in that they are the first that can be both stabilised through freeze-drying, and formulated to ensure that they can be recovered through re-hydration without neutralisation by the action of bile acids in the gut.

Principles of freeze-drying
Freeze-drying is a well-established and standard process used in the pharmaceutical industry to stabilise drug products, with associated benefits (increase in shelf life, ease of transport, smaller load sizes, fewer temperature controls).
There are three main stages in the complete drying process: freezing, primary drying and secondary drying.

Freeze-drying process variables include
• Degree of shelf contact e.g. tray/no tray, container shape.
• Cooling rate and ultimate freezing temperature and effect on ice crystal growth
• Time, temperature and pressure of each step of the sublimation process
• Efficiency of heat transfer - conduction and convection, container material.
• Radiative heating (fewer vials exposed to side-wall radiation in larger freeze dryers) scale up/optimisation.
• Annealed or non-annealed - ice crystal size, pathways for vapour to escape.
• Extent of secondary drying (desorption) to achieve low residual water level.

Variables in the excipients/active material
• Material type/structure e.g. amorphous or crystalline, material complexity
• Critical temperature of formulation, based on the formulation itself but sometimes with an element of process-dependence (e.g. rapid vs. slow cooling)

Successful freeze-drying relies on choosing suitable components for the formulation and establishing the critical temperatures for the product.

Freeze-drying is frequently used for bacteria but is not yet commonly used for probiotic bacteria with the exception of some starter cultures in the dairy industry that are freeze-dried.

The project

To produce stable L. crispatus (a challenging organism) efficiently, which can be delivered successfully to the gut, three organisations worked on the three stages of the project:

1. Microbial Developments Ltd worked on the fermentation stage, with the aim of  optimising conditions to give a >20% increase in harvest compared to current production methods.
2. BTL worked on the formulation and freeze-drying stage to select protective agents and develop freezing and drying conditions to give less than a 1 log reduction in viability and good storage stability at ambient temperature.
3. Professor Nigel Slater at the Department of Chemical Engineering and Biotechnology at the University of Cambridge worked on the gut delivery stage to develop and test a number of petrochemical-derived and natural Bile Adsorbing Resin (BAR) reagents for co-encapsulation with the bacteria.

Finally these stages were brought together to produce a commercially viable stable probiotic product in capsule form.

Within the fermentation stage the variables studied included:
• Culture medium components
    o Modification by addition of    a  range of adjuncts
• Physical parameters
    o Temperature
    o pH
• Point of harvest comparisons
    o Early log phase
    o Late log phase
    o End log phase
    o Stationary phase

The optimum combination of harvest time, media constituents and physical parameters gave >20% improvement in viable counts over historical batches and scaling this up to a 1200L fermentation batch gave a further improvement in harvestable yield.

Within the formulation and freeze-drying stage the variables studied included:
• Bulking/protective agents
    o 12 candidates examined
    o Compared with absence of protective agent
    o Method of addition (dissolving into slurry versus mixing slurry with concentrated solutions)
• Freeze-drying conditions
    o Cycles based on collapse temperature of each formulation, with a view to making cycles as efficient as possible

Less than a 1 log reduction in viability was achieved using a standard food excipient on a 6-day cycle.

Bile adsorbing Resins
The BAR reagent stage variables studied included:
• Eight petrochemical and naturally occurring BARs (from renewable sources) that were compared against positive and negative controls
• BARs that were subjected to a simulated bile solution for sixty minutes to assess extent and rate of degradation
• The three best-performing BARs were then combined with freeze-dried powders and dosed into enteric coated capsules for degradation and stability studies.

Using a combination of different BAR reagents provided protection of L. crispatus against simulated bile acid.

Project results
All project aims were successfully achieved. The probiotic bacteria were freeze-dried, bile acid stabilisers added and all material encapsulated. Accelerated stability tests were carried out, with early data indicating promising levels of bacterial survival:
• Less than a 1 log drop observed after 14 days at 37°C
• More than 50% viability observed after 14 days at 25°C

Next steps and implications for the industry
While the optimised variables will probably differ in detail for each probiotic organism, the processes developed through the project can potentially be applied to any organisms whose use creates practical difficulties of poor stabilisation and formulation.
The production of a dosage form of probiotic that can ensure higher levels of surviving bacteria reaching the gut will enable the benefits of probiotics to be optimally exploited through new and more profitable supply chains, thus accessing wider market opportunities. It will also allow more efficient utilisation of resources through lower wastage from extended stability and product shelf life.

The next steps of the project include applying the same development process to other (high value, low yield) bacteria requiring stabilisation and/or delivery to the gut in a viable state using an oral capsule dosage form, and taking 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.

Further information
Biopharma Technology Limited:
Technology Strategy Board:
Microbial Developments Limited:
The University of Cambridge:

Contact information
Dr Kevin R. Ward
Biopharma Technology Limited
Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK
Tel. +44 (0)1962 841 092
Fax +44 (0)1962 841 147

Sarah Webster is a free-lance scientific writer.


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