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Bioencapsulation innovations and technologies

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A. BACKGROUND

The first application of microcapsules was proposed by Chang in 1964. The method was based on the interfacial polymerisation. The reaction of an aqueous diamine with organic acid dichloride at the interface of a 1/2 "water in oil" emulsion is carried out under severe conditions. A milder and simple method of immobilisation was developed in England (Kierstan and Bucke, 1977) and was the main starting point for cell encapsulation. By dropwise addition of sodium alginate solution into a calcium chloride solution, hydrogel beads are formed. The method is biocompatible (neutral pH, ambient temperature, physiological osmotic pressure).

The applications of microcapsules in Biotechnology

Many biological systems in their natural state are immobilised. Without retention, cells would be washed away by water flow. Most functions of living systems are based on the confinement of reactions in a limited space. The membrane insures also the protection of the internal material.

Many biotechnological processes need to be carried out using immobiliation of the biocatalysts. Encapsulation is considered as a powerful method of immobilisation. There are many examples of applications of this technique in different fields:

- soil inoculation is often unsuccessful because cells are washed out by rain. Cell encapsulation allows the maintenance of continuous inoculation and reaches ten times higher cell concentrations,

- plant cell cultures allow production of different metabolites used for medical, pharmacological and cosmetic purposes. Cell immobilisation improves the efficiency of the cultures by imitating cell natural environment,

- immobilisation seems to be the technique of choice in many industrial processes in food and especially in beverage production. The technologies such as beer, wine, vinegar and some others already experience some immobilisation approaches traditionally with adhesion culture (i.e. acetobacter in vinegar production) and a modern approach with entrapment of yeast biomass (i.e. sparkling wine),

- continuous fermentation allows higher global performances than batch fermentation. To avoid wash-out of the biological catalyst from the reactor, it is necessary to immobilise it. This principle is applied in ethanol and solvent production, sugar conversion or waste water treatment.

It appears that bioencapsulation has a strong potential in most biotechnology fields and especially in agriculture and food.

The diversity of the bioencapsulation methods

Encapsulation by interfacial polymerisation and by alginate gelation represent two main axes of research in biomolecule immobilisation. Other methods described in literature are presented below.

In interfacial polymerisation, the diamines may be replaced by materials of divers structures (e.g. proteins, polysaccharides and chitosan). Operating conditions may then be modified to pH~7 without use of toxic solvents. Transacylation allows avoiding the use of toxic cross-linking agents. The encapsulation of cells by a mild interfacial chemical process then becomes possible.

Alternatives to alginate gel have been proposed. Most of them are based on thermal gelation. The beads are produced through dispersion of a sol-gel in vegetal oil and gelified by lowering the temperature. A commonly used material for entrapment is Kappa-carrageenan, more stable than alginate beads in biological media. But the high temperatures (45 to 50(C) used in the process are unfavourable especially for immobilisation of plant and animal cells. Consequently more than 80% of cell encapsulation processes is still carried out using alginate as the matrix for immobilisation.

Hydrogel beads provide the immobilisation of the cells but not protection. Alginate beads may be coated by simple suspension of the alginate beads in the polylysine solution. A 10 to 30 mm membrane is formed around the beads. The conditions for the formation of this membrane are very mild. Chitosan has been proposed to replace PLL but most research is still based on PLL, especially for encapsulation of animal cells and artificial organs.

More recently, immobilisation inside a membrane was obtained by dropwise addition of the alginate solution into chitosan solution (interfacial coacervation). A membrane, very similar to the previous case, will be formed at the interface. Dautzenberg used also semi-synthetic materials. The use of strongly charged polymers in place of weakly charged ones allows the formation of improved membranes.

The remaining questions

Only selected examples of encapsulation methods are listed above. However, after more than 30 years of intensive research on the subject, there are still some shortcomings:

- alginate beads as matrices represent more than 80% of cases of cell immobilisation,

- the limited selection of food grade carriers and especially its shelf life and inert structure in relation to substrate and to bioprocess products that is taking place in this direction is very important and has to be considered very carefully,

- when alginate beads are coated, the coating material is mainly restricted to poly-L-lysine,

- most bead-forming operations are based on the syringe-drop method which is not suitable for large scale processes,

- relatively little knowledge has been acquired about the microcapsule characteristics.

Many researchers and industrialists with interest in the field agree today that the success of the bioencapsulation is largely restricted by the above limitations. However, implementing new bioencapsulation methods and processes requires vast interdisciplinary knowledge and expertise and consequently collaboration among laboratories is essential.

B. OBJECTIVES AND BENEFITS

The main objective of the Action is to develop the bioencapsulation methods in view of their transfer and development in agricultural and industrial applications. On the other hand, the action aims to foster cooperation in Europe, in part through yearly meetings and scientific contribution to international conferences, in research and development of bioencapsulation technologies, in order to:

- increase the awareness of possibilities offered by the new materials in the biocatalyst encapsulation,

- identify new processes allowing use of these materials under mild and biocompatible conditions,

- collaborate to develop and test the encapsulation processes on a large scale,

- characterise and optimise the microcapsule materials and the related processes to suit the requirements of specific applications in biotechnology, agriculture and nutrition,

- disseminate the acquired knowledge among the EU scientific community involved in bioencapsulation,

- evaluate the different applications of biocapsules in terms of economy, social impact, technological limitations, feasibility, potential partners and so on. Such evaluation will be diffused to the scientific and industrial communities to allow better selection of useful research programs and industrial development,

- identify industrial partners and involve them in the Action. This is particularly true for SMEs, which have been created in the last years. The Action will help them in developing their activities. Moreover, the Action has the objective to promote creation of new SMEs, based on the identification of potential markets.

The proposed project has, as a secondary objective, the development of bioencapsulation methods for medical (i.e. artificial organs), pharmaceutical (e.g. vaccine delivery) and cosmetic applications (e.g. protection of active compounds). The development of these technologies will be also beneficial for bioencapsulation applications in agronomy (e.g. seed inoculation), food sciences (e.g. probiotic delivery and food fermentation) and environmental biotreatment (e.g. combined anaerobic/aerobic fermentation).

The research on microencapsulation is two-fold: designing the encapsulation technology and its application. However very few collaborations exist between process designers and users. Many encapsulation methods have been designed without considering the elementary rules of application in a specific field. On the other hand, some users apply encapsulation methods without sufficient knowledge and control of the technology. In many cases, experimental approach has not taken into account the scaling up of the process.

C. SCIENTIFIC PROGRAMME

The outline of the research project is as follows:

Working Group I: New materials

WG1 will focus on selection and characterisation of material suitable for bioencapsulation. This part includes a better characterisation of actual material and determination of their optimum conditions of uses.

1. selection, synthesis, purification of material

The encapsulation processes have been developed around a limited number of natural polymers. Alginate is by far the most usual material, even if it is recognised that alginate beads are not stable enough for industrial applications. Many other materials have been tested but their application remains very limited.

Natural polymers present serious advantages for low cost applications but for specific applications, they require purification. Synthetic polymers allow better control of the properties but are generally expensive. An intermediate solution would consist in semi-synthetic polymers such as cellulose sulphate. It is therefore proposed:

- to make an inventory of natural polymers able to form either gel beads or microcapsules, and to define correct procedure for their purification,

- to evaluate the production of synthetic or semi-synthetic polymers able to form either gel beads or microcapsules,

- all these polymers will be investigated to compare their capacity to form quickly and easily, strong and stable capsules, in regard to alginate beads,

- they will also be evaluated for cost, availability (natural polymers) and large scale production.

2. characterisation (molecular properties, rheological characterisation)

Most researchers, involved in alginate bead applications, are mainly concerned with the behaviour of the encapsulated material but not by the encapsulating material itself. Professor Skjak-Braek (Norway) has shown that selection of alginate types has very strong impact on the capsule properties (stability, homogeneity, biocompatibility). The report on this study has changed the attitude of many researchers in the handling of alginate. The selection of polymers retained above will then be investigated for their solution properties:

- molecular weight (mean and distribution),
- functional group distribution and strength,
- rheology of their solutions,
- gelification behaviour,
- relations of these properties with the microcapsule forming capacity.

Working Group II: Development of bioencapsulation processes

WG2 will study the different processes of bioencapsulation and will determine the critical parameters to optimise them. It will link its observation with the selection of materials. It will also define characteristics of microcapsules in function of both process conditions and material selection.

1. gelation, polymerisation, coacervation, coating.

Many methods of bioencapsulation exist. However, only a very limited number of them have a real impact. For the non specialist, it is very difficult to select a method and get enough information to apply it in optimised and controlled conditions. This explains why most groups have limited their research to alginate beads. Even for this material only external gelation, a mainly non-scalable process, is used. Therefore, to serve the scientific and industrial communities, it would be useful:

- to test different technologies, with the above selected materials, including:

- ionic and thermal gelation,
- interfacial polymerisation and cross-linking,
- interfacial coacervation and coextrusion,
- ionic and spray coating,
- to compare the methods in terms of simplicity, biocompatibility and scaling,
- to evaluate resulting capsules on the basis of the methods described below,
- to create a comprehensive handbook of the bioencapsulation methods.

2. characterisation of the microcapsules (size, mechanical resistance, permeability).

The industrial development of bioencapsulation depends on the possibility to provide clear and complete information about the properties of microcapsules. For example, the design of a reactor using encapsulated material is linked to the microcapsule properties. Agitation and aeration will be limited by the microcapsule mechanical resistance. Fluidised bed liquid velocity must be calculated in function of the microcapsule settling velocity. Microcapsule material must be selected to avoid liquefaction in fermentation media.

However, the tools for determining easily and efficiently the microcapsule properties are lacking. Often, several methods are required to describe adequately the microcapsule behaviour. It is therefore proposed to optimise and standardise methods to evaluate:

- size and size distribution,
- mechanical strength,
- stability in application media,
- permeability to low substrate and impermeability for biocatalysts.

Working Group III: Evaluation of the bioengineering parameters:

The objective of WG3 will be to evaluate scaling-up rules and different aspects of the industrialisation of microcapsules. At this stage, it would be especially interesting to involve industrial aspects in the Action.

1. optimisation of the beads dispersion process

All bioencapsulation methods may be divided in two main steps. In the initial step, the material to be encapsulated must be divided in fine droplets. In a second step, the droplets are solidified by gelation or membrane formation around the droplet.

In the case of dispersion, two methods may be applied, i.e. extrusion and emulsification. Extrusion allows small production of relatively monodispersed droplets. Reduction of the droplet size requires the application of a force on the droplet (air flow, electric potential, etc.). However, the energy dissipation leads to size distribution. To maintain low size dispersion and to control the mean size, a careful analysis of the drop break must be performed. Emulsification allows really larger scale but results in large size dispersion. It requires physicochemical understanding of the driving parameters to optimise the process. The focus of the working group will be:

- to analyse the drop breakage processes,
- to test and to optimise different methods of extrusion,
- to adapt emulsification methods for viscous and non-newtonian solutions (generally the case in encapsulation),
- to develop devices for laboratory scale that may be used for standardisation of methods between groups.

2. rules of process scaling-up

In most cases, the production of biocapsules is limited to millilitres of capsules. Some technologies were proposed to scale-up bioencapsulation methods, such as multivibrating nozzles, vibrating spinning discs, for emulsification using static mixer. However, these technologies have not been completely developed due to lack of collaboration between researchers from the bioencapsulation field and engineers.

As much as possible in collaboration with the manufacturers, we will investigate:

- different scale-up devices for extrusion,
- continuous production of emulsion using static mixer,

and test them for different encapsulation technologies taking into account the cost and the adequation of the system with the industrial requirement.

Working Group IV: Compartmentation of the immobilised systems

WG4 will be in charge of investigating the behaviour of the biocatalysts while immobilised inside a microcapsule. This part includes the influence of encapsulation on the biocatalyst but also consequences of encapsulation on the performance of the overall process (fermentation, soil inoculation level).

1. biocatalysts in microcapsules (viability, integrity, kinetics).

Studying biocapsules independently of the included biological functions would have no sense. Taking into account the range of applications, some simple models must be established, allowing the evaluation of the impact of encapsulation on the biocatalyst behaviour.

It is important to realise research which will generate a knowledge of general interest. Too much research in the field was restricted until now to single systems without the possibility of extrapolating the data. It is also important that this part of the Action will emphasise advantages (or disadvantages) of the encapsulation in comparison to other immobilisation technologies.

The working group will then evaluate different methods and material of encapsulation taking into account:

- viability of (microbial, plant and animal) cells and enzymes,
- integrity of the biocatalysts and their behaviour at long term,
- biocatalyst activity (growing, pathway),
- and define rule of selection in function of the type of biocatalysts.

2. microcapsules in situ (fermenter, implantation)

As was shown in the previous paragraph, the systems will be investigated with regard to the internal components. Obviously, the behaviour of the external medium is important as well. We can divide it into two categories: in vitro and in vivo.

A classical in vitro system is fermentation. The working group will need to answer the questions:

- how capsules will behave in a bioreactor, especially on a large scale?
- how capsules will behave in a natural environment such as soil?
- what type of reactors will be suitable for using biocapsules?
- how to adapt an actual reactor to be used with encapsulated materials?
- how will the use of encapsulated material affect downstream processing?

Working Group V: Assessment of the capsules in function of the applications

WG5 will have the task of unifying all the information obtained by Working Groups I to IV and defining rules to apply bioencapsulation realistically and efficiently to agriculture and industrial applications. This includes correlated classifications of microencapsulation technologies, biocatalysts and applications and defining the resulting rules.

The aim of the Action is to generate fundamental and practical knowledge, useful in the development of new bioencapsulation applications. The applications have to be classified in function of their specific requirements. Some biocatalysts may support some levels of stress without loss of activity. Mechanical resistance enhancement must not introduce mass transfer problems linked to strong membranes.

Such analysis will promote a larger spread of microencapsulation technologies. For example, interfacial polymerisation is generally considered as too toxic for biocatalyst encapsulation. However, 90% of final activity has been found with urease. On the other hand, interfacial polymerisation allows the production of capsules with a very strong but very thin membrane (200 mm), as compared to most other techniques (5 to 30 mm).

The final objective of the Action is to draw up an "encyclopedia" of bioencapsulation technologies as a result of the proposed collaboration. The handbook will include information on:

- how to achieve encapsulation,
- how to characterise the microcapsules,
- how to industrialise the process,
- how to optimise the biocapsule applications.

D. ORGANISATION AND TIMETABLE

The organisation and coordination of the Action will be undertaken by the management committee, assisted by the COST secretariat, in accordance with the common procedures for COST initiatives. The management committee will be composed of coordinators from each working group, representing national interest and official scientific representatives of each country participating to the Action.

The general program is divided into three phases. The first one concerns the acquisition of data useful to define a generic framework of the Action (2 years). The second phase is devoted to the application of the data to different models (1,5 years). In the last one the correlation of the data will be realised to allow development of pre-industrial processes (1,5 years). The total duration of the action is estimated to 5 years.

The five Working Groups are defined as developed in the scientific program. It must be pointed out that the fifth working group consist to compile and exploit the information acquired in the other working group. It will be then composed of the sub-committees of the first four working groups. Each working group will have a chairman and a co-chairman to follow the progress of the group's studies, to organise working group meetings and to encourage the mobility of the researchers between the institutes involved with the groups. They will check that the links and the communications are in place between the different working groups, an important element for the success of the COST Action. Each year they will draw up a summary of the studies undertaken by their group. The relations between Working Groups are described below.

The planning meeting will be organised with all participants of the COST initiative in order to set up the working groups and elect chairmen and co-chairmen. A chairman meeting will clarify precisely the general organisation and links with the COST secretariat and plan.

Two group meetings per year during the life of the working group (see table below) are scheduled: one independent and one in parallel with the annual meeting. Working group meetings will enable a presentation of study advances, the analysis and resolution of obstacles found, to suggest movement among the research groups and to harmonise the methods used.

The annual meetings will allow for links between the different working groups, to check that studies for each group fit properly into the common objectives of the COST Action.

Management committees will be scheduled on the basis of two per year as a preamble to the annual and mid-year meetings. They will be responsible for collecting the summary of the study results from each group and putting them together in an interim report.

The final seminar will summarise all the work of the working groups and will underline any advances achieved towards the objective defined. It will be edited for a scientific publication in the form of a booklet (different editors have shown significant interest in microencapsulation), and/or be published on the internet for a large spread of the information.

Working Groups:

1. new materials (including selection, synthesis and characterisation)
2. development of bioencapsulation process
3. evaluation of the bead dispersion process (including scale-up)
4. biocatalyst behaviour inside the beads
5. assessment of the capsules in function of the applications

Training:

During the COST Action, there will be evaluated and promoted divers initiatives linked to training of "young scientists" (students and those from less-well-developed countries) such as:

- exchanges of young scientists between laboratories through bi-national and/or Erasmus programs,

- support of workshop organisations in the field and sponsoring of young scientists to attend to these workshop and important meetings in the related field (International workshop on Bioencapsulation, European Biotechnology Conferences),

- organisation of summer schools (such as Euroconferences),

- development of specific diplomas (the applicant is negotiating the creation of a French "Diplome de Recherche Technologique" on microencapsulation to be extended to a European applied master later),

- shared technological platforms will be promoted to allow scientists to be initiated into diverse aspects of bioencapsulation.

E. ECONOMIC DIMENSION

The national costs have been estimated from data provided by 23 groups which have actively participated in the preparation of the Action or otherwise indicated their interest. However, while preparing this project, more than 200 research groups have been identified in the COST geographical area. In all cases, information is only related to the part of the groups involved in bioencapsulation.

The Action has encountered interest from, and will be performed in relation with, industrialists. However, industrial background (research and production), has not been included because of the difficulties of getting useful information.

Country Professors PhD Budget
scientists Postdocs (ECU)

Belgium 8 14 1 250 000
Czech Republic 1 4 250 000
France 6 11 1 220 000
Germany 6 10 1 400 000
Greece 2 4 400 000
Ireland 7 11 1 195 000
Italy 3 9 625 000
Netherlands 1 3 250 000
Portugal 2 3 300 000
Slovakia 2 2 260 000
Spain 6 8 1 100 000
Switzerland 6 7 965 000
UK 3 4 750 000

TOTAL of the national costs per year 53 90 10 465 000.

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