Info

Helm

(as Soares & Mota, 1997)

Stratford

(as Soares & Mota, 1997)

Miki et al.

(as Smit et al., 1992)

Recover cells

Centrifugation

Centrifugation

Centrifugation

Wash

EDTA (250 mM)

EDTA (250 mM)

Water

Resuspend in buffer

Sodium chloride

(250mM), pH 4.5, 1 x 10s cells/ml

(a) Deionised water, 4 x 109 cells/ml

(50 mM), pH 4.5

Sodium acetate (50 mM), pH 4.5, OD620 = 2.5, 30 minutes 'acclimatisation'

Calcium concentration

4mM

4mM

9mM

Final volume

25 ml in measuring cylinder

40 ml in 100 ml conical flask

0.65 ml in 1 ml cuvette

Mix/agitate

18 inversions

Orbital incubation at 120 rpm

Whirlimix (20 seconds), invert cuvette (5 x )

Incubation time

< 7 minutes

< 4 hours

5 minutes

Aliqouts removed

0.2-1 ml from '20 ml'

Stop shaking, after 30 seconds remove 0.21 ml from just below the meniscus

No

Cell count

Disperse in sodium chloride (250 mM) and determine OD62o

Disperse in EDTA (250 mM) and determine OD62o

Monitor decrease in od620

An interesting paper that seeks to define more precisely the conditions necessary for flocculation (and by implication for a 'test') is that of van Hamersveld et al. (1996). Here, flocculation was investigated in situ, in fermenter at the end of fermentation. Using some innovative techniques, factors such as medium composition, calcium concentration, pH, temperature and shear rate (both laminar and turbulent) were evaluated in terms of floe size, settling rate, bond strength and fraction of single cells. This work 'adds value' in demonstrating the implications of changing environmental variables, and, importantly, notes that temperature, which is frequently ignored, is ideally 15°C (van Hamersveld et al., 1996).

An important contribution to the methods debate is the detailed work of Soares and Mota (1997) who compared their absorbance-based version of the Helm test with a composite Stratford method. Essentially, the two methods yielded results that where indistinguishable in terms of flocculation or, indeed, precision although the Helm method was significantly quicker to perform than the Stratford approach. Soares and Mota (1997) plump for their variant of the Helm test as a 'rigorous and an objective method for quantification of yeast flocculation'. So perhaps despite earlier pessimism, the possibility of an agreed standard test is now not so far away.

4.4.7.2 New approaches. After more than 50 years of wrangling and development of the Burns test, it is timely to consider the opportunities for the development of alternative approaches to assess flocculence. The advent of 'bead'

technology perhaps provides new routes to the isolation and separation of floccu-lent cells.

Although it is not clear whether or not cell surface hydrophobicity is a universal factor in flocculation (see Section 4.4.2.6), bead technology has been applied successfully to its measurement (Wilcocks & Smart, 1995; Rhymes & Smart, 1996) and isolation (Straver & Kijne, 1996). Essentially hydrophobic cells adhere to hydrophobic beads, either 1-2 (im polystyrene-coated latex (Straver & Kijne, 1996) or 0.8 (im latex (Wilcocks & Smart, 1995). The larger beads of Straver and Kijne (1996) are paramagnetic, enabling their removal with a magnet.

A similar approach that potentially offers better targeting of cells with the capability to flocculate is the use of 'Dynabeads' (Anonymous, 1996a). These superparamagnetic polymer particles (2.8 (im) can be coated with ligands that enable selective isolation of cells with a magnet. This is because the beads are coated with streptavidin, a protein with a strong affinity for the B vitamin, biotin. Ligands such as antibodies, proteins, lectins, sugars or nucleic can be attached after being tagged with biotin ('biotinylated'). Once customised, the beads can then be used to probe suspensions for target cells, which are then selectively recovered via a magnet (Fig. 4.43).

Fig. 4.43 Diagrammatic representation of lectin magnetic bead technology.

Preliminary studies suggest that customised Dynabeads may be useful to probe or quantify flocculence. Unpublished work (Wendy Box, Andrew Prest and David Quain) has shown Dynabeads with an attached snowdrop lectin to bind to brewing yeast cells with almost 100% efficiency (Fig. 4.44). This lectin binds to mannose and, presumably, interacts with the cell wall mannose receptors. In agreement with Stratford (1993), receptors appear to be constitutive, as binding was effectively 100% throughout fermentation. Unfortunately attempts to probe flocculence lectins with biotinylated dimannose on beads have been unsuccessful. It is assumed that although only two mannose residues are necessary for lectin binding (Section 4.4.6.2), bio-

Fig. 4.44 Attachment of magnetic beads to flocculent yeast.

tinylation results in some sort of steric hindrance. It is anticipated that trimannose may be a better substrate for biotinylation and subsequent bead-cell binding. In the event of Dynabeads being able to probe for flocculence lectins, it is anticipated that a more functional flocculence test will be devised.

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