MICROENCAPSULATION IN YEAST CELLS – STRUCTURE AND FUNCTION: A CRYO-SEM APPROACH S. C. Duckham†*, A. Burgess‡, L. Hinds† and P. Echlin§.
†Micap plc, Ashton House, 1 The Parks, Lodge Lane, Newton-Le-Willows, Merseyside, WA12 0JQ. ‡University of Cambridge, Multi Imaging Centre, Department of Anatomy, Downing Street, Cambridge, CB2 2DY. §Cambridge Analytical Microscopy, 65 Milton Road, Cambridge CB4 1XA. *To whom correspondence should be addressed. E-mail –
[email protected] ABSTRACT Introduction Encapsulation of chemicals in microorganisms was established as a patented technology in the late 1980’s (AD2 Ltd. 1987). Since then moves have been made to commercialise the technology in Europe and the USA, initially as a flavour delivery encapsulation process, by companies including Corn Products Inc/ Bestfoods and Micap plc (formerly Fluid Technologies plc).
may not only be an affect on the release characteristics but also on agglomeration, dispersion properties, the maximum level of encapsulated active or the efficiency of encapsulation. Cryo-scanning electron microscopy (SEM) was carried out; images detailing the changes in yeast structure, the interaction between yeast surfaces and essential oils and comparative data on proprietary yeasts will be presented.
The technique for encapsulating essential oils and other chemicals in Bakers Yeast (Saccharomyces cerevisiae) was demonstrated by the presence of droplets found within the 4-5 micron yeast capsule using confocal microscopy and transmission electron microscopy techniques by Bishop et al. (1998). These studies indicated that a high degree of membrane structural integrity remained following the encapsulation process. The aim of this current paper was to identify the structural characteristics of the yeast cell before, during and after the encapsulation process. The yeast is subject to various processes which may affect the encapsulation and release performance of the end product. In ethanol production, for example, the yeast by-product is inactive. In contrast, yeast produced as a primary product for bread making or the brewing/wine industry has a short growth cycle and may be presented as “cake” or as a dry form following mild drying conditions such as extrusion for pelleting processes. In those cases the yeasts, to varying degrees, are active.
Experimental Yeast types investigated. Torula yeast (Candida utilis; Bakers/Brewers yeast (Sacch. cerevisiae), instant dried active powder and moist cake for breadmaking and as a dried powder by-product of ethanol bio-fuel production.
Although the manufacturing process of microencapsulation using yeast has now been developed on the commercial scale, the mechanism of release in situ is not fully understood. Imaging using electron microscopy is presented here as a tool to investigate structural changes which arise from yeast processing and during different stages of encapsulation. It is clear that the source of yeast has a great impact on the performance of encapsulates. This
Encapsulation of essential oils. Typically an essential oil such as orange peel oil or tea tree oil was mixed for several hours with water, dried yeast powder or moist cake. The mixture was separated by centrifugation and the re-suspended pellet was spray dried. In one experiment, at specified times, 1, 10, 20, 30, 40, 50 minutes, 1, 1.5, 2, 3, 4 and 5 h, a 2 ml sample of the slowly agitated yeast (Sacch. cerevisiae)/Tea Tree (Melaleuca alternifolia) oil (R C Treatt & Co Ltd., UK) and water suspension was removed and centrifuged to recover the yeast cells. A small piece of the moist yeast pellet was used for microscopy and the remainder was retained for chemical analysis. Analysis involved preparing ethanol extracts of wetted samples which were filtered and analysed by GC-FID. Typically samples were prepared and analysed in triplicate. Electron microscopy. Images were taken of the raw material yeast cake, following dilution with water (1:1) and at times throughout the internalisation process. All samples were prepared for low temperature SEM in the following way.
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Immediately following the preparation of encapsulates approximately 1mm3 of material was quench cooled and a clean frozen fracture face was prepared then sputter coated with gold:palladium alloy. The scanning electron microscope was an FEI Company XL30 Field Emission Gun instrument, with a resolution of 1nm. Specimens were examined and photographed at 140K, 10-6 torr, 5keV and 5-25pA.
Encapsulation of essential oils takes place rapidly in the early stages and approaches a maximum after 5 hours of incubation (figure 4). Particles, initially only appearing on the membrane surface, appeared to undergo swelling. Following the addition of essential oil, particles resembling droplets appeared in the cytoplasm.
Results and Discussion Initial studies included cryo-SEM analysis of fresh yeast cake (figure 1), active dried powder (figure 2) and other commercially available yeast (figure 3).
Figure 5 Droplet distribution throughout the cytoplasm and across the cell wall surface 2 hours after the start of the encapsulation process.
Figure 1. A cryo-SEM image from a proprietary bakers yeast sampled from an active “cake” showing organelles.
These droplet-like structures were absent from the nucleus. Previous studies have demonstrated a loss in viability during encapsulation (Bishop et al 1998) and the presence of essential oil throughout the cell may have been the cause of this.
Figure 2. A cryo-SEM image from an instant dried active bakers yeast following re-hydration showing the membrane surface.
Conclusions There are clear differences in the cell wall surfaces between yeast of different type, for example Bakers Yeast and Torula Yeast, from different commercial sources, and as a result of different processing techniques. The ability to produce clearly resolved images of internal cell contents using cryo-SEM was adversely affected by the degree of yeast processing. A high degree of cellular damage was noted in products repeatedly spray dried.
Figure 3. Cryo-SEM images of some proprietary yeasts used for encapsulation. Products are typically rinsed with water and spray dried for storage purposes. a) Sacch. cerevisiae from ethanol bio-fuel production following repeated washing and drying. b) Yeast (Sacch. cerevisiae) for bread making following washing and drying.
c) Torula Yeast (Candida utilis) used in animal feed, processed using a roller drying technique.
Figure 4. A time course for the encapsulation of Tea Tree Oil in active Bakers Yeast cake (duplicate samples).
Changes during encapsulation occurred following the dilution of cake samples with water. Within 10 minutes small bright particles on the surface of the membrane appeared to more than double in diameter to around 50 nm. Bright particles 12-15 nm in diameter have been viewed in yeast in previous studies and identified as proteins (P. Echlin, pers. comm). Further changes followed the addition of essential oil. Essential oil appeared to be associated with these sites and over a 2 hour period appeared on the surface of the plasmalemma and throughout the cytoplasm. Ribosomes were identified in studies by Walther and Müller (1999) as particles up to 25 nm in size distributed throughout the cytoplasm, however, these were darker particles found only within the cytoplasm. Further studies are planned to confirm the presence of essential oils as the droplet-like structures found in the yeast cells in this paper.
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References AD2 Ltd, 1987, European Patent 0 242 135 assigned to Bestfoods, US. Bishop, J. R. P., Nelson, G., and Lamb, J., (1998), Microencapsulation in yeast cells. Journal of Microencapsulation, 15, (6), 761-773. Walther P., & Müller M, 1999. Biological ultrastructure as revealed by high resolution cryoSEM of block faces after cryo-sectioning. Journal of Microscopy, 196, 279-287.
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