WO2004064971A2

WO2004064971A2 - Process for preparing microcapsules having an improved mechanical resistance

Info

Publication number: WO2004064971A2
Application number: PCT/CH2004/000029
Authority: WO
Inventors: Anne Peters; Ian Marison; David Serp
Original assignee: Inotech AG
Current assignee: Inotech AG
Priority date: 2003-01-23
Filing date: 2004-01-20
Publication date: 2004-08-05
Anticipated expiration: 2005-07-23
Also published as: WO2004064971A3

Abstract

A process for preparing microcapsules having an improved mechanical resistance, more specifically monodisperse microcapsules consisting of an organic liquid core surrounded by a hydrogel polymer membrane comprises: a) using selected hydrophobic organic liquid material as core constituent; b) using selected acrylamide and/or acrylamide derivatives together with alginate as constituents of the monomer mixture leading to the surrounding polymer membrane; c) initiating and then performing polymerization of the selected monomers up to the desired cross-linkage level of the polymer membrane; and d) subjecting the core and the membrane components to a suitable microcapsulation technology, whereby the microcapsules thus achieved have an improved mechanical resistance. Such microcapsules are useful e.g. as vectors for active compounds or as extraction means.

Description

PROCESS FOR PREPARING MICROCAPSULES HAVING AN IMPROVED MECHANICAL RESISTANCE

FIELD OF THE INVENTION

The invention refers to a technique suitable for in situ product recovery, more specifically recovering or extracting lipophilic compounds from aqueous medium.

BACKGROUND OF THE INVENTION

Hydrophobic liquid core capsules are widely used in the perfume and cosmetic industries for the encapsulation of aromas and solvents and in agriculture to decrease herbicide volatility and hazards associated with their application . Such capsules are usually produced by coacervation, emulsion, or spraying techniques and are characterized by a wide size distribution. Biotechnological applications are limited to the use of hydrophobic core capsules for in- situ product recovery (ISPR) in a technique termed capsular perstraction (WO 00/73485). In this technique an organic phase, dibutyl sebacate, was surrounded by a calcium alginate hydrogel, to form liquid- core capsules. Such capsules survived autoclaving, although this was accompanied by an important loss of mechanical resistance. The placing of the capsules directly within a bioreactor, or as part of an external recycle loop, was used to successfully extract 2- phenylethanol from a yeast bioconversion process, thereby overcoming respiratory inhibition by both phenylethanol and dibutyl sebacate. However, this application was severely limited by the rapid loss of mechanical resistance during incubation in the yeast culture medium, with the result that capsules burst, releasing dibutyl sebacate into the medium, which resulted in inhibition of yeast metabolism. Thus, while the potential of capsular perstraction was demonstrated, further development of the technique was limited by the stability of the capsules to culture conditions, such as the ionic composition of the medium and mechanical shear stresses. For efficient mass transfer the surface area to volume ratio of the capsules should be high and the capsules have a uniform size. Of the currently available techniques for the production of microcapsules the prilling technique, an extrusion method based on laminar jet break-up, which involves a concentric two-fluid nozzle shows the most promise (Brandenberger and Widmer 1997). However, the limitation of the prilling, and other extrusion- based methods, is the need for a polymer solution which is sufficiently viscous to allow the formation of spherical capsules, while not so viscous as to prevent jet break- up, together with a rapid polymerisation reaction. For these reasons alginate complexation with calcium ions has frequently been used. However calcium alginate gels have a very poor mechanical stability in the presence of monovalent cations and chelating agents, which are usually present in biotransformation media (Serp, Catana et al. 2000).

SUMMARY OF THE INVENTION

The purpose of the present invention is to propose new and useful extraction means, more particularly liquid-core microcapsules which prove efficient in e.g. capsular extraction of inhibitory products from bioprocesses or bioconversions.

Therefore, there has been developed a new process for the production of microcapsules composed of a hydrophobic liquid core surrounded by a cross-linked hydrogel polymer membrane which exhibit a significantly improved mechanical resistance when compared to similar prior known microcapsules. These liquid-core capsules may be used e.g. in capsular extraction for the removal of inhibitory products from bioprocesses and bioconversions. They have, among others, the advantage of having a high surface area to promote rapid mass transfer, while separation of the organic core phase from the aqueous environment by the capsule membrane. As first object of the invention there is a process for the preparation of monodisperse microcapsules consisting of an organic liquid core surrounded by a hydrogel polymer membrane, which comprises

a) using selected hydrophobic organic liquid material as core constituent ; b) using selected acrylamide and/or acrylamide derivatives together with alginate as constituents of the monomer mixture leading to the surrounding polymer membrane ; c) initiating and then performing polymerization of the selected monomers up to the desired cross-linkage level of the polymer membrane ; and d) subjecting then the core and the membrane components to a suitable microcapsulation technology, whereby the microcapsules thus achieved have an improved mechanical resistance.

As another object of the invention there is a method for vectorizing nutriments, perfumes, flavours, chemical reactants, enzymes, markers or the like, which comprises subjecting the selected ingredient to the above mentioned process while adding said ingredient to the core component prior to any encapsulation reaction or while mixing achieved microcapsules with the selected active ingredient until core saturation.

Still a further object of the invention comprises a method for recovering lipophilic compounds from an aqueous medium which comprises preparing separately microcapsules according to the above mentioned process, adding the achieved microcapsules to the aqueous medium, performing extraction up to the desired extraction rate and eventually recovering loaded microcapsules from that medium.

In a preferred embodiment of the invention acrylamide and N-hydroxymethylacrylamide monomers were chosen for the preparation of the capsule membranes together with alginate in order to achieve the desired properties obtention of spherical capsules using the prilling technique.

In a preferred embodiment of the invention, the micro capsulation technology which is applied is the laminar jet break-up co-extrusion technique, although any technique allowing proper polymerization speed monitoring and an easy control of microcapsules dispersion can be applied.

In still another preferred embodiment of the invention the resulting cross-linked material (microcapsules) is further treated with complexing reactants for removing divalent cations, like e.g. calcium cations, from the hydrogel microcapsule membrane. Doing so results in an ever larger increase of the mechanical resistance of the microcapsules.

Capsules having membranes composed of a copolymer of acrylamide and N- hydroxymethylacrylamide exhibited even higher mechanical stability towards chelating agents.

So there are now means for producing monodisperse liquid- core microcapsules with a higher mechanical stability, as compared with those composed of an alginate membrane, and a hydrophobic liquid core. Such capsules should be resistant t e.g. for capsular perstraction and for the immobilization of biocatalysts.

DESCRIPTION OF THE INVENTION

Materials and methods

Chemicals

Stock solutions of 40% (w/v) acrylamide(AA) and N, N'-methylene-bis- acrylamide(MBA) in a ratio of 19/1, N, N'-methylene-bis-acrylamide(IVIBA), Hydroxymethylacrylamide(NMAM) and Tert-butyl hydroperoxide (70% in water) were obtained from Sigma (Buchs, Switzerland). Acrylamide (purity >99.5%), sodium pyrosulfite(Na₂S₂O₅) and dibutyl sebacate were obtained from Fluka (Fluka Chemie AG, Buchs, Switzerland). Alginate LVG (Batch n°005-281-03) was supplied by Pronova (Lysaker, Norway).

Preparation of capsules Capsules composed of a hydrophobic liquid core and a hydrogel membrane were prepared using the co-extrusion jet- break- up technique. The encapsulator (Inotech Encapsulator I EM) was fitted with a concentric nozzle with an internal diameter of 200μm and an external diameter of 300 μm or an internal diameter of 400μm and an external diameter of 500 μm. Two syringe pumps (200 series, kd Scientific, Boston, USA) were connected to the encapsulator to supply dibutyl sebacate (oil) through the central nozzle and polymer solution through the external nozzle. Spherical capsules were obtained by the application of a vibrational frequency with defined amplitude to the co- extruded jet and collected in a gelling bath placed 18 cm below the nozzle and agitated by a magnetic stirrer (length 4cm). Polymer flow rate, oil flow rate and vibration frequency were empirically determined for the different polymer compositions and for the different nozzles used.

A stock solution of sodium alginate (7%) was prepared in Tris/HCI, pH 7 and filtered through a 0.2Dm filter under a pressure of 4-6 bar. Different stock solutions of acrylamide /methylene-bis-acrylamide were prepared and filtered (Steritop 0.2Dm, Millipore, corporation δOAshby Road Bedford MA 01730-2271). These solutions contained (a) 38%AA, 2% MBA; (b) 38% AA, 4%MBA; (c) 38% AA, 8%MBA; (d) 38% AA, 10%MBA; (e) 47,5%AA, 2,5% MBA; (f) 53,2% AA, 2,8% MBA. The stock solutions of alginate and acrylamide were then combined, with agitation, to a ratio of 1:1 in order to prepare solutions with the desired concentration. To 10 ml of these polymer solutions were added 20μl Tert-butyl hydroperoxide The polymer/monomer solution was then co-extruded with the oil, dibutyl sebacate, into the gelling bath composed of 8% CaCI₂, 20mM Tris/HCI, pH7, 1% Tween 80 and 0.1-0.4% Na₂S₂O₅. The resulting capsules were incubated in the gelling bath for 45min, filtered and washed extensively with de-ionised water to remove any un- reacted reagents.

Sterilization of capsules

A batch of capsules were incubated in a citrate solution (20g/l) for 1 hour, filtered and re- suspended in fresh citrate solution, followed by autoclaving (Zirbus HST/32) at 121°C for 20min.

Measurement of capsule size distribution

The size and size distribution of capsules was determined using a microscope (Zeiss Axiolab, Switzerland) fitted with a video camera (CCD-IRIS, Sony, Japan) interfaced to a PC operating with the Cyberview (Cervus International, Courtaboeuf, France) image analysis software. A sample of 60-200 capsules was examined and the mean standard deviation determined.

Measurements of mechanical resistance

The mechanical resistance of the capsules was measured using a Texture Analyser (Model Ta-XT2I, Stable Micro Systems, England) as the mean force (gram) necessary to break one capsule.

An applied compression speed is 0.1 mm/s was used. The mechanical resistance values are presented as the mean of 20 different measurements. Results and discussion

The production of liquid- core microcapsules using the prilling technique has been described previously (Stark 2001).

In the present work stabilization was attempted by adding a covalent structure into the ionically cross-linked hydrogel, using acrylamides, or derivatives of acrylamides, copolymerized with bis-acrylamide. Traditionally ammonium persulfate is used as an initiator and N,N,N',N'-tetramethylethylenediamine(TEMED) as the activator. However, this system does not allow a sufficiently rapid rate of polymerization to enable the formation of monodisperse spherical capsules using extrusion systems, such as the jet- break- up technique. Consequently, different redox couples were attempted in order to increase the rate of polymerization. One of these, composed of tert-butyl hydroperoxide as oxidizing agent and sodium metabisulfite (pyrosulfite) as reducing agent, was used to form a pair of radicals, to initiate the polymerization at ambient temperature. For alginate concentrations of 3.5%, spherical capsules could be obtained, due to the increased viscosity limiting exo- diffusion of the acrylamide.

As a result all further experiments were performed with a constant alginate concentration of 3.5%, while varying other parameters, such as monomer concentration, monomer type, initiator concentration and size. The concentration of CaCI₂ in the gelling bath was also maintained constant at 8%. Preliminary experiments (not shown) indicated that lower concentrations resulted in slow gelation, leading to an important level of leaching of acrylamide, whereas concentrations above 8% showed no improvement. The surfactant Tween 80 was added to the gelling bath solution in order to decrease the surface tension, thus facilitating the complete wetting of the capsules and formation of spherical capsules.

The different empirical parameters such as dibutyl sebacate flowrate (F_DBS), polymer flowrate (F_poi_ymer) and frequency were determined empirically for different polymer solutions. Using the jet- break- up technique the droplet diameter is mainly dependent on the nozzle diameter and vibrational frequency applied, with smaller capsules obtained at higher frequencies. The frequency applied is itself dependent on the nozzle diameter, rheology and surface tension of the polymer solution, however, no mathematical description has been found which fully describes the relationship between these parameters for concentric nozzle systems(Heinzen 1995). Earlier work (Stark 2001) indicated that the flow rate of the polymer solution (alginate) has to be at least double the flow rate of the hydrophobic liquid in order to encapsulate the oil. However, this ratio may be much lower when acrylamide is added to the alginate solution. This is probably due to the lower surface tension of the polymer mixture (52mN/s for alginate 3.5%, AA 23.75%, MBA 1.25%) compared with the alginate solution (72mN/s for alginate 3.5%).

Effect of cross-linker concentration

Cross- linking density is a key factor controlling the size of the membrane pores, the pore volume fraction and the interconnections, therefore the influence of the cross- linker content was studied by varying the MBA concentration between 1% and 5% and keeping the concentration of acrylamide (19%) and alginate (3.5%) constant, with an initiator concentration in the gelation bath of 0.1%. An upper limit of 5% MBA was chosen since at higher concentrations spontaneous polymerization occurred and no capsules could be formed. The mechanical strength was measured for capsules stored in water and compared with similar capsules stored in a solution of trisodium citrate (20g/,l pH 8.0) at 20°C. Citrate acts as a complexant with a high affinity for calcium ions. As a result capsule membranes composed of non- chemically cross- linked calcium alginate would be expected to solubilize, or swell significantly, upon incubation in citrate solutions. When cross- linked acrylamide/ alginate capsules were stored in a citrate solution the stability was found to vary as a function of the cross- linker (MBA) concentration. Thus the mechanical stability of capsules produced using 1% MBA actually increased by 72% (86 g/capsule) compared with similar capsules stored in water (50 g/ capsule). On the other hand for higher concentrations of MBA the mechanical resistance decreased to a value of only 10 g/capsule (86% decrease compared with storage in water) with an MBA concentration of 5%.

In the presence of high concentration of MBA, it is therefore clear why alginate contributes to the mechanical strength of the gel, since the pores are large and allows the gel to have a denser and less brittle structure. Treating such gels with citrate, results in solubilization of the alginate, some of which diffuses through the large pores of the membrane into the surroundings, and leads to a very fragile macroporous membrane. This could clearly be demonstrated by addition of CaCI2 to the citrate solution, and observing the formation of gels/ precipitates due to reaction of Ca2+ with alginate leached from the capsules. Under certain conditions the membrane porosity is such that the core oil can actually diffuse out of the capsules.

The addition of PEG to the gelling solution creates constraints, which restrict the movement of polyacrylamide chains. As a result, during the gelling process, the growing chains of polyacrylamide are forced to aggregate via hydrogen-bonding prior to cross-linking (Righetti 1995; Righetti and Gelfi 1996). In the present study alginate gels rapidly in contact with Ca2+ in the gelling solution, thereby retaining acrylamide and allowing sufficient time for radical polymerization to occur and a macroporous capsule membrane to be formed.

The membrane formed with a cross-linker concentration of 1% is more homogeneous than membranes formed from higher cross-linker content. Therefore when exposed to chelating agents, alginate is partially released from the membrane. The latter rapidly re-orients to arrive at a new equilibrium. The presence of sodium alginate in the membrane results in additional swelling because of the osmotic driving force, however the covalent cross-linkages will oppose this swelling, leading to an elastic membrane retraction force.

Effect of acrylamide monomer concentration

The earlier results showed that capsules from 3,5% alginate, 19% AA and 1% MBA exhibited the highest mechanical stability. Consequently, further characterization of capsules was undertaken by maintaining this composition, with the ratio of MBA/total monomer constant at 5%, and varying the total monomer concentration between 20 to 28%. Solutions with a total monomer concentration above 28% could not be studied due to the occurrence of spontaneous polymerization.

Mechanical stability of capsules made from 20-28% total monomer concentration was tested after incubation in water, after citrate treatment and after thermal sterilization at 121 °C for 20 min in the presence of citrate (20 g/l). The results (Figure 6) show that the mechanical strength of capsules, made from the range of total monomer concentrations studied, did not change significantly when stored in water, regardless of the treatment applied. However, when similar capsules were stored in citrate (20 g/l) there is an increase in mechanical strength for all total monomer concentrations with a distinct optimum for a concentration of 25%. At this concentration the mechanical strength increased by between 30-40% compared with capsules stored in water (Figure 6). This increase is probably due to the formation of more homogeneous gels since the lower the concentration of monomer during gel formation the more heterogeneous the gel obtained (Sayil and Okay 2001). Swelling is observed for all total monomer concentrations when capsules are incubated in citrate and after thermal sterilization, although the swelling is more important for lower concentrations of acrylamide, probably due to a lower density of cross-linking.

Effect of initiator concentration Since it was shown that an increase in the total monomer concentration resulted in capsules resistant to thermal sterilization, it would be expected that increasing the initiator concentration would increase the level of polymerization of acrylamide, since the rate of polymerization is dependant on the square root of the initiator concentration and the monomer concentration (Thomas and Wang ). High initiator concentration increases the rate of polymerization, resulting in a membrane containing higher levels of polyacrylamide, leading to capsules with improved mechanical stability when incubated in citrate and thermally sterilized.

In the case of larger capsules (r_ext, 0,50 mm), the mechanical strength of capsules stored in citrate increases with increasing initiator concentration (0.1 % to 0.4%) to attain a value of over 140 g/capsule with 0.4% initiator. This increased stability was retained after thermal sterilization, particularly at higher initiator concentrations. This is in accordance with the observations made earlier of stability as a function of total monomer concentration in which capsules prepared from the highest monomer concentration showed the highest stability to thermal sterilization.

Effect of chelating agents and pH

Since the liquid- core capsules produced in this work were intended for use in the in- situ extraction of compounds from biotransformation processes, it is essential that they are stable in buffer solutions, such as phosphate, over a wide pH range. In order to test this capsules made from alginate (3.5%), AA (23.75%) and MBA (1.25%) were incubated in phosphate buffer (0.1M) at different pH values over 2 to 24 hours and the mechanical resistance determined.

The results show that the mechanical strength of the capsules increased within the first two hours of incubation in phosphate buffer of pH between 6 and 9, to values nearly three fold higher than those obtained during storage over the same time period in water. Furthermore the capsules retained this stability over the 24h period tested.

Influence of the type of monomer

Acrylamide monomer contains an unsaturated reactive amide group. As a result it can be derivatized to form other compounds that contain further reactive groups, such as N-(hydroxymethyl)-acrylamide or methylolacrylamide (NMAM) which, while less reactive then acrylamide, have the advantage of having N-hydroxymethyl groups available for self-crosslinking (Warson 1990) and which may also be used for the immobilization of enzyme or cells (Krysteva, Shopova et al. 1991; Yildiz, Isik et al. 2001).

Initial experiments were performed with a polymer/monomer solution containing NMAM (23.75%), MBA (1.25%) and (3.5% ) alginate. The resulting capsules dissolved immediately upon incubation in citrate solution (20g/L). Changing the polymer/monomer ratio and addition of AA to form solution containing NMAM (12%), AA (11.75%), MBA (1.25% ) and alginate (3.5%) resulted in capsules with a mechanical resistance (300 g/ capsule) more than two-fold higher than those obtained from acrylamide-methylene- bis- acrylamide (120 g/capsule). The results shown are for an initiator concentration of 0.4% only, since such capsules were stable over the whole 24 h period tested, whereas capsules produced with 0.2% initiator exhibited a similar resistance for the first 20 h but subsequently lost this resistance over longer incubation periods.

Capsules formed from NMAM (12%), AA (11.75%), MBA (1.25% ) and alginate (3.5%) could be sterilized for 20 min at 121°C in a citrate solution without any loss of mechanical strength nor measurable swelling . The reason for this thermal stability is that the principle reaction taking place when polymers containing NMAM are heated is the formation of formaldehyde, due to the self cross- linking NMAM. This self cross-linking reaction may also explain the higher mechanical strength of capsules formed in presence of hydroxymethylacrylamide (Warson 1990). Capsules produced with an initiator concentration of 0.4% show no measurable swelling throughout the different processes, such as incubation in citrate and thermal sterilization.

Monomers of acrylamide and hydroxymethylacrylamide are more hydrophilic and less reactive than alginate, and will tend to diffuse towards the external surface of the capsule membrane whereas alginate will tend to accumulate around the hydrophobic core. At the same time, the initiator will diffuse from the external part of the capsule membrane to the core, leaving more primary molecules in the external region of the membrane. Since MBA has more than twice the reactivity of AA or NMAM, due to the presence of two vinyl groups, it will react faster. Consequently the polymer formed earlier will be more cross-linked than that formed later. The highly cross-linked polymer will be located in the external region of the membrane, whereas loosely cross-linked molecules will predominate at the inner surface region of the membrane, forming a network structure. When capsules are treated with citrate, the alginate within the membrane will solubilize and accumulate to form an aqueous liquid membrane surrounding the hydrophobic liquid core. Table legends

Tablel : Parameters for the extrusion of solutions of alginate and acrylamide using the jet- break- up technique. Abbreviations: F_DB_S, flow rate of dibutyl sebacate, Fpoiymer flow rate of polymer solution; AA, acrylamide; NMAM, hydroxymethylacrylamide; MBA, N, N'- methylene- bis- acrylamide.

Improvements to the mechanical stability of two-phase liquid-.core capsules produced by the prilling technique has been achieved. In the absence of chelating agents, higher concentrations of cross-linker improves greatly improves the mechanical strength of two-phase capsules composed of cross-linked polyacrylamide/alginate membranes, whereas in the presence of chelating agents the capsules resist solubilization although mechanical resistance is reduced. On the other hand addition of an optimum concentration of MBA (5%) (g MBA/g total monomer) leads to capsules with a high mechanical resistance when exposed to chelating agents at pH values between 4 and 9, as well as to thermal sterilization.

A hydrogel membrane composed of a copolymer of acrylamide and derivatives of acrylamide, such as hydroxymethyl acrylamide, exhibits even higher mechanical resistance. It should also be possible to use alternative derivatives of acrylamide, with other functional groups, to produce capsules using the same system.

It should also be possible to use thermosensitive co- polymeric membranes using thermosensitive monomers such as ispropylacrylamide copolymerized with acrylamide or derivatives of acrylamide. Thermoresponsive hydrogels of N- isopropylacrylamide-N-hydroxymethylacrylamide have previously been synthesized by redox- polymerization for enzyme immobilization (Yildiz, Isik et al. 2001).

Latter the polyacrylamide membrane might be used as a support for enzyme immobilization in reactions where there is a need to remove the product into an organic phase because of limitted solubility in water or inhibition problems. The spherical porous capsules have the advantage of a high surface area available for enzyme attachement and for fast extraction.

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Claims

1. - Process for the preparation of monodisperse microcapsules consisting of an organic liquid core surrounded by a hydrogel polymer membrane, which comprises

2. - Process according to claim 1, wherein the hydrophobic organic liquid material is an oil of natural or chemical origin or a water immiscible organic solvent or a mixture of same, e.g. dibutyl sebacate, oleic acid or fractionated coconut oil, an aliphatic or aromatic hydrocarbon like e.g. hexadecane or toluene, a middle or high molecular weight alcohol, aldehyde, ketone or ester.

3. - Process according to claim 1 , wherein the acrylamide or acrylamide derivative is selected from acrylamide, methylene-bisacrylamide and acrylamide derivatives having a further reactive group such as N-(hydroxymethyl)-acrylamide or methylolacrylamide.

4. - Process according to claim 1 , wherein the polymerization is initiated by means of an initiator suitable for achieving a sufficiently high rate of polymerization to enable formation of monodisperse spherical microcapsules in the conditions of reaction, more specifically redox couples able to initiate polymerization at room temperature.

5. - Process according to claim 1 , wherein at least one of the selected monomers is a thermo sensitive monomer such as isopropylacrylamide.

6. - Process according to claim 1 , wherein the cross-linked material resulting from the polymerization is treated with a complexing reactant for removing divalent cations from the hydrogel microcapsule membrane, like e.g. sodium citrate.

7. - Process according to claim 1, wherein the microcapsulation technology which is applied to the core and membrane components is laminar jet break-up co-extrusion technique.

8. - Microcapsules consisting of an organic liquid core surrounded by a hydrogel membrane having an improved mechanical resistance obtainable by means of the process according to claim 1.

9- Microcapsules according to claim 8, useful as vectors for active compounds like nutriments, perfumes, flavours, chemical reactants, enzymes, markers or the like.

10. - Microcapsules according to claim 8, useful as extraction means, namely for in situ product recovery, preferably recovery of lipophilic compounds from an aqueous medium.

11.- Method for vectorizing nutriments, perfumes, flavours, chemical reactants, enzymes, markers or the like, which comprises subjecting the selected ingredient to the process of claim 1 while adding said active compound to the core component prior to any encapsulation reaction or while mixing achieved microcapsules with the selected active compound until core saturation.

12.- Method for recovering lipophilic compounds from an aqueous medium which comprises preparing separately microcapsules according to the process of claim 1 , adding the achieved microcapsules to the aqueous medium, performing extraction up to the desired extraction rate and eventually recovering the loaded microcapsules from that medium.