Disruption of bakers’ yeast using a disrupter of simple and novel geometry
Introduction
The disruption of microbial cells is an important unit operation in the preparation of intracellular products from micro-organisms. There are a number of ways in which this may be achieved, and they may be based on mechanical action, e.g. homogenisers and bead mills, or non-mechanical action, e.g. freezing and osmotic shock [1]. The mechanical methods are generally seen as most appropriate for large scale disruption, with homogenisers being commonplace. Homogeniser pumps typically operate at pressures in the region between 50 and 120 MPa and function by pumping liquid through a spring-loaded ring seal, e.g. APV Gaulin Homogeniser [2], [3]. At these operational pressures it is normal for the cell suspension to require several passages through the homogeniser to obtain maximum or acceptable disruption. There are several disadvantages to multiple passes, the most important being that cooling is required between the passes as the energy released during the operation is sufficient to raise the temperature by up to 20°C in some cases. Increased operational pressures, although increasing cell disruption, result in greater temperature rises and heat transfer then becomes a critical consideration. Finally, most homogenisers that are available are not easily adapted for effective containment, a particularly important aspect if the cells utilised involve rDNA techniques.
A new type of commercial cell disrupter manufactured by Constant Systems Ltd. (Warwick, UK) has several advantages over most other disruption systems in that it is sterilisable, is capable of a high level of containment and may be cleaned in place. The homogeniser is capable of operation at very high operating pressures of up to 275 MPa (40 kpsi), these pressures being achieved when pumping the process liquid through a small jet (0.1–0.2-mm diameter) using a hydraulically driven piston. It is able to process quantities from 20 to 30 ml to several litres in prolonged runs (at flow rates over 200 ml/min) using a single pressure head. The novel breakage system (the disruption head) is capable of utilising high operational pressures in a controlled manner and the simple geometry of this system is anticipated to allow an analysis to be made of possible mechanisms of disruption. A research program has been initiated in association with the manufacturers to assess the effectiveness of the homogeniser for disrupting micro-organisms. We report here some of this work and show that the system is very effective in disrupting baker's yeast. We also investigate some of the factors that control the efficiency of the disruption.
Section snippets
The constant systems cell disrupter
The Constant Systems Series B disrupter is a single head system with all its internal drillings and external fittings designed for full containment. It has points for sample inlet, product outlet, seal drain, cleaning fluid and steam input. The machine is fitted with pipework, control valves and control system (internal PLC) to enable simple and effective cleaning and sterilisation cycles. A hydraulic pump and accumulator (housed below the high pressure head) generate the high pressure fluid
The effect of operating pressure on cell disruption
A set of experiments was conducted to assess the effect of operating pressure on the protein release from baker's yeast. In these, a series of 100-ml samples of yeast suspension containing 30 g/l of yeast (dry weight) in 50 mM phosphate buffer were passed through the disrupter at pressures ranging from 50 to 275 MPa. The disrupted samples were then assayed for the optical density, the protein released and the G6PDH activity. Table 1 shows some properties of the homogenates as a function of
Discussion
The Constant Systems cell disrupter was very effective at disrupting baker's yeast, being able to release over 85% of total cell protein in a single pass. The main reason for this was that it could operate at high pressures without causing unacceptable damage to the macromolecules in the lysate. The design of the low pressure chamber with its integral cooling jacket means that the excessive temperatures normally associated with such high operating pressures were avoided, it being possible to
Acknowledgements
We would like to thank John Harbidge of Constant Systems Ltd. for valuable discussions and his company's financial support. Also, we wish to acknowledge the BBSRC support for some of this work (CPY), the EPSRC (GMC) and that of the EU for ERASMUS exchange funds (CA).
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