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Fig. 7.16 Iodine staining of (a) yeast cells and (b) yeast slurries (Quain & Rubb, 1983. with permission from the Institute of Brewing). Intensity of brown staining is proportional to glycogen content. (This figure is repeated in the colour section)

Fig. 7.16 Iodine staining of (a) yeast cells and (b) yeast slurries (Quain & Rubb, 1983. with permission from the Institute of Brewing). Intensity of brown staining is proportional to glycogen content. (This figure is repeated in the colour section)

would have a reduced requirement for wort oxygenation for standard fermentation performance. This would be detectable by monitoring sterol levels in pitching yeast. A reasonably rapid method for yeast sterol determination has been developed (Rowe el al., 1991). This relies on a change in the absorbance spectrum of the polyene antibiotic filipin, which occurs after it reacts with sterol. Of course, the necessity to monitor sterol, glycogen and possibly trehalose on a routine basis begins to stretch the concept of a rapid inexpensive vitality test!

The intracellular concentration of ATP can be used as a measure of condition. Thus, viable cells must generate sufficient ATP for energy maintenance during periods of starvation. ATP levels in yeast cells (Hysert el al., 1976) are conveniently monitored using bioluminescence (see Section Manson and Slaughter (1986)

determined the correlation between ATP content and fermentation performance in samples of yeast that had been stressed by storage at high temperature prior to pitching. A good correlation was obtained, although no better than that seen with methylene blue viability staining. A more accurate reflection of the energy status of a cell is that which takes into account the ratio of concentrations of ATP and ADP to AMP, in other words the concept of 'adenylate energy charge' (Chapman & Atkinson, 1977). Under normal conditions cells maintain a high adenylate energy charge (> 0.75). A low value may be taken as evidence of stress. However, because cells tend to maintain high values for this parameter, a significant decrease is indicative of cells that are close to death, and this would probably be apparent by other simpler tests, such as a viability stain.

The redox state of the cell is of critical influence to many aspects of yeast metabolism, not least glycolysis and ethanol formation. A manifestation of redox is the relative concentrations of NAD/NADH. The intracellular concentrations of these metabolites may be determined by fluorescence spectroscopy. As Lentini (1993) pointed out, the cost of the apparatus for such determinations would probably mitigate against its application for routine testing.

Variable pitching yeast physiology may arise in ways other than in response to environmental stimuli. For example, the mean cell age of the yeast could be a variable factor, perhaps related to the number of generations of fermentation the yeast had passed through, or a particular cropping regime (see Section The age of yeast cells is related to the number of bud scars present on the cell surface. Bud scars consist of the carbohydrate chitin (see Section that does not occur elsewhere in the envelope. Chitin can be detected by specific dyes such as primulin and calcofluor such that the extent of staining reflects cell age. Measures of cellular activity. Another approach to assessing yeast physiology is to measure some aspect of metabolic activity, preferably one that reflects behaviour during fermentation. Several vitality tests of this type have been proposed. The most obvious and direct predictive fermentation test is to perform a laboratory fermentation trial, using the type of apparatus described in Section 5.9. Such tests have the advantage of providing information regarding most of the parameters of interest in a real fermentation and similar wort may be used. Unfortunately, the time required to undertake the test would be too long for routine application. Instead, other aspects of the activity of yeast may be measured which relate to fermentation performance. Possible parameters are rate of oxygen uptake, rate of evolution of carbon dioxide, exothermy and rate of ethanol formation (Boulton & Quain, 1987; Daoud & Searle, 1987).

Measurement of the specific oxygen uptake rate forms the basis of the BRi yeast vitality apparatus, the use of which has been described by Kennedy (1989). It consists of an accurately attemperated water bath in which a predetermined quantity of yeast suspension is placed. The specific rate of oxygen uptake is measured using an integral Clark-type electrode with output to a recorder. Results were presented by Kennedy (1989) who indicated that differences in this parameter could be detected in production pitching yeast samples, which were not evident from a simple viability test. Specific rates of oxygen uptake are related to yeast sterol content (see Section 3.5.1).

Another vitality meter has been developed by Muck and Narziss (1988) which measures carbon dioxide formation as increase in pressure in a sealed container. Like the BRi vitality meter, readings are taken whilst a known quantity of yeast is held under controlled conditions. Results are obtained within one hour. Similar measurements may be made with a Warburg manometer or an automated respiro-meter (Mathieu et al., 1991). Fluorometric vitality tests. As discussed in Section, several tests of yeast viability have membrane function as their basis. There is the assumption that viable cells will have an intact and functional membrane, whereas non-viable cells will have total loss of membrane function. The disparity between results of tests such as methylene blue staining and slide or plate counting techniques suggest that conditions of membrane competence which lie in between total collapse and fully functional are probable. The fact that sterol concentration in membranes is a known variable, which is influenced by environmental factors, confirms this view. Vitality tests based on limited fermentation rely on the yeast membrane being functional but only in part. Measurement of the specific rate of oxygen uptake more closely probes membrane integrity because of its relation to sterol concentration. Another useful membrane probe is the ability of yeast cells to acidify the external medium, both spontaneously and in response to a supply of exogenous glucose, termed the acidification power test.

Acidification power is defined as the sum of the spontaneous pH change determined after suspending yeast cells in water and the substrate-induced pH change after addition of glucose to the suspension (Sigler et al., 1980,1981a, b, 1983; Opekarova & Sigler, 1982; Sigler & Hofer, 1991). The observed changes in pH largely reflect the activity of the membrane- bound H +ATPase. Thus, the cell extrudes protons in order to assist with the control of intracellular pH and to maintain a proton electrochemical gradient across the plasma membrane (see Section This gradient supports active transport of nutrients into the cell, and therefore it is essential to the maintenance of viability and is intimately related to metabolic activity during fermentation. Spontaneous acidification is a function of the energy status of the cell and positively correlates with glycogen content. Glucose-induced acidification relates to both membrane state and glycolytic flux. Some counterbalancing activities also occur which have the opposite effect on the external pH. Proton influx occurs as a counter ion in some transport processes and passive proton influx takes place if the membrane becomes de-energised. It has been suggested that this latter effect may be a symptom of ethanol toxicity (Fernanda Rosa & Sa-Correia, 1994). The magnitude of acidification is significantly influenced by univalent cations in sugar-metabolising yeast. Kotyk and Georghiou (1994) reported a 20-fold increase in acidification rate in S. cerevisiae in the presence of potassium ions. These authors concluded this was due to an effect of this cation on reactions producing ATP and not the membrane-bound H +ATPase.

An acidification test suitable for application for testing pitching yeast condition was developed by Kara et al. (1988). The test is a modification of that of Opekarova and Sigler (1982) in which notably the incubation temperature was changed to 25°C. A known wet weight of yeast is washed in chilled distilled water by repeated suspension and centrifugation. The test commences by suspending the washed yeast pellet in a known volume of distilled water at 25°C. The slurry is incubated with constant stirring for 10 minutes during which time the pH is monitored at intervals of one minute. The change in pH after 10 minutes' incubation is taken as the spontaneous acidification power (AP10). After this time, glucose solution was added to the suspension to give a final concentration of 0.1% w/w. The pH was monitored for a further 10 minutes. The change in pH during the second incubation is the glucose-induced acidification power (AP2o). Using this procedure, the authors demonstrated in laboratory studies an inverse straight-line correlation between acidification power and fermentation performance, measured as time to half gravity. However, they noted that it was unreliable when used with acid washed yeast.

Fernandez et al. (1991) applied the AP test at commercial scale and concluded that it was predictive of fermentation performance. In this study the deleterious effects of storage of pitching yeast under inappropriate conditions were identified and it was observed that in addition to the relation with fermentation rate, a correlation was observed between acidification power and VDK stand-times. Mathieu et al. (1991) measured acidification power on samples of yeast removed from fermenter at intervals after pitching. They observed a sharp drop in AP10 during the first few hours of fermentation. This was attributed to the rapid drop in yeast glycogen content associated with the aerobic phase of fermentation. They reported that the correlation between acidification power and fermentation performance (measured as decrease in gravity, "Plato per day) was better if the acidification power was applied to yeast 24 hours after pitching. Accordingly, they modified the test to include a pre-treatment step in which pitching yeast was exposed to wort for 15 minutes prior to acidification measurement. This resulted in a decrease in total acidification power (decrease in APio, slight increase in AP2o) which was shown to result from an interaction between the yeast cell surface and a trub component, possibly a tannoid. Providing the pre-treatment was performed, these authors also reported a good correlation between acidification power and subsequent fermentation performance.

Others have sought to modify the acidification power test in different ways. Patino et al. (1993) suggested that it would be better to convert each pH reading into the corresponding proton concentration and sum the differences for each of these over the time course of the test. This provided a parameter related to the magnitude of the pH change, which they termed the 'cumulative acidification power'. This corrected for the non-linearity of pH change during the acidification power test. In addition, they proposed that the test was made more reliable by substituting maltose for glucose on the basis that the latter formed the major sugar in worts. Furthermore, since changes in the concentrations of ions other than H + occur during the test, it would be more meaningful to measure conductance, as opposed to pH.

Iserentant et al. (1996) pointed out that a drawback of the AP test was its relative insensitivity with 'high vitality' yeast. Thus, because of the logarithmic basis of the pH scale, the maximum acidification power is limited to about 2.8. These authors obviated this problem by modifying the procedure such that the pH was maintained at a constant value by titrating with 0.1 M sodium hydroxide. The volume of sodium hydroxide needed to do this was measured and this was referred to as the titrated acidification power. It was demonstrated that this test was useful for yeasts such as those grown under aerobic conditions which would be predicted to have a very high

AP. Conversely, it was not sensitive enough for low vitality yeast, and in this case, it was suggested that conductivity was a more useful measure than pH.

Another of the many effects of yeast metabolism that may be observed during fermentation are changes in extra- and intracellular metal ion concentrations. Mochaba et al. (1996) noted that fluctuations in the concentrations of cations such as potassium, calcium, zinc and magnesium during fermentation were dependent on yeast condition. They developed this into a vitality test based on the release of magnesium ions, which occurs when yeast is pitched into wort (Mochaba et al. 1997). The test involves suspending a small known quantity of washed yeast (0.1 g wet weight) into 10 ml of fresh brewery wort. After one minute contact time a small sample of the suspension is removed and filtered to remove yeast. The magnesium concentration in the filtrate is determined using a commercial spectrophotometric kit. In laboratory and plant trials, it was shown that a positive correlation existed between yeast vitality and magnitude of magnesium release. Yeast judged as being highly vital by this method produced superior fermentation performance in all respects. Thus, more rapid attenuation, greater ethanol yields and shorter VDK stand-times. The differences detected by the magnesium release test were not evident from simple viability testing.

Many of the methods of viability determination that use fluorochromes can also be extended to provide information regarding physiological condition. Imai et al. (1994) developed a procedure using a derivative of fluorescein, 5 (and 6) carboxyfluorescein. This procedure involved liberation by esterases of fluorochrome in viable cells. A vitality test was developed that involved measurement of intracellular pH based on a calibration curve that related fluorescent intensity using a range of buffers of differing pH values. Data was presented that indicated a correlation between intracellular pH of a number of yeast samples and fermentation performance based on rates of sugar consumption. The method was claimed to be superior to the acidification test in that it was capable of distinguishing differences over a greater range of physiological conditions. Thus, a positive correlation between results of the vitality test and fermentation performance was observed for yeast samples with intracellular pH values up to 5.7. However, in a data set of 555 production yeast samples, more than 90% had an intracellular pH greater than 5.7, and therefore would be indistinguishable by the acidification test.

The potential of fluorochromes as monitors of yeast physiological condition can be properly unlocked if used in conjunction with flow cytometry (Donhauser et al., 1993; Edwards, 1996; Hutter, 1997). Flow cytometric tests are rapid and since all cells within the sample population are examined there is no requirement for testing a defined quantity of cells. A wide range of fluorochromes is available which allow many aspects of physiology to be examined. These include viability, membrane competence, intracellular pH, distribution of genealogical age, ploidy, phase in cell cycle and others. Not only is it possible to identify the proportions of cells in a given state within the population but with sophisticated cytometers these sub-populations may be separated and collected for further analysis. Vitality tests - a summary. The key element of any vitality test is that it must provide information that can be acted on by the brewer. The potential for influencing the outcome of the brewing process is likely to reflect the effort and cost expended on the test. In this respect, vitality tests may fulfil a number of roles. At their simplest, they will be used to judge batches of pitching yeast on a reject quality limit basis. For the majority of brewers, a viability test will serve for this function. The methylene blue haemocytometer counting method, or other simple microscopic approaches, are equal to this task. The fact that methylene blue staining may significantly over-estimate viability compared to plate counting methods in grossly deteriorated yeast is irrelevant in this case since no brewer given the choice would seriously contemplate use of such yeast. Fluorescent staining techniques more accurately reflect the viability as judged by a colony forming unit method; however, there is a significant additional cost as a fluorescent microscope is needed.

Vitality tests may be used to monitor long-term drift in factors that influence yeast physiological condition, and hence fermentation performance and beer quality. In this case, any drift that resulted in gross deterioration of pitching yeast, would be signalled by the viability methods and again no additional testing would be warranted. However, the change may be of a type that influences yeast physiology without affecting viability. In addition, inherent variability in raw materials contributing to wort composition and inconsistencies in fermentation control and yeast handling can result in subtle variations in yeast physiology which are not detectable by simple viability testing. In these cases additional assessment may be justified.

Of the many tests that have been described, acidification power offers many advantages. It has all the requirements of a routine QA test in that it is rapid, simple, inexpensive and uses readily available laboratory equipment. Most importantly, it is predictive of subsequent fermentation performance such that it allows selection of an appropriate pitching rate. In its original incarnation, it is a less useful test for discriminating yeast samples of very 'high' vitality. The modified acidification tests, such as titrated acidification power, address this problem without the need for much greater outlay in terms of laboratory equipment.

Undoubtedly assessing yeast physiology by flow cytometry is a very powerful and flexible tool, which has the potential to deliver the most detailed analysis. Unfortunately at present this comes at considerable cost. For this reason, this approach is unlikely to find use as a routine method until costs fall by at least an order of magnitude. Until that time, flow cytometers will be destined to be confined to the research laboratory.

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