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Fig. 6.20 Effect of pitching rate on fermentation rate (shown as time to half gravity) and yeast growth extent (weight of total crop minus weight of pitched yeast) for pilot scale (8 hi) high-gravity lager fermentations (Boulton & Box, unpublished data).

In a brewery fermentation, the inoculum is large, typically a quarter to a fifth of the total crop. In this situation, the proportion of nutrients assimilated by each cell in the inoculum becomes significant. If the size of the inoculum (pitching rate) is increased, this ratio decreases and the effect becomes more significant such that total growth extent is restricted by nutrient availability.

It follows from the data shown in Fig. 6.20 that selection of the optimum pitching rate involves a trade-off between fermentation rapidity and loss of efficiency due to the proportion of wort sugars used for new biomass formation. Whilst it is true that very high pitching rates would give both short fermentation times and low growth, this option is precluded because of unacceptable shifts in flavour due to the limited yeast growth. Thus, low yeast growth extent results in elevated levels of esters. From a practical standpoint, if yeast growth is very restricted, insufficient yeast may be available for re-pitching. In addition, the quality of the crop may be reduced because of the relatively high proportion of cells derived from the pitching yeast.

The patterns of yeast growth rate and extent are further modulated by the oxygen concentration supplied to the wort (Fig. 6.21). Here is shown the effect on attenuation rate of varying both pitching rate and initial dissolved oxygen concentration in a high-gravity lager fermentation. Increase in both parameters resulted in faster fermentation rates such that the most rapid rates were obtained with high pitching rates and high dissolved oxygen concentration.

The effects on yeast growth of simultaneously varying pitching rate and dissolved

Fig. 6.21 Effect of varying pitching rate and initial dissolved oxygen concentration on fermentation rate (shown as time to half gravity) for pilot scale (8 hi) high-gravity lager fermentations (Boulton & Box. unpublished data).

oxygen are complex because, depending on the values, different nutrient limitations may result. Where oxygen is limiting, the pattern of growth versus pitching rate is as shown in Fig. 6.20. At any given pitching rate, as the oxygen concentration is increased, more growth is permitted and the peak is moved towards the right until growth becomes limited by some other component of the wort. At different pitching rates the balance between limitation by oxygen or another nutrient is altered and different patterns of growth result.

The effects of varying pitching rate and oxygen concentration on the formation of volatile flavour components are also complex. The concentrations of esters formed during fermentation are influenced by the nature of the nutrient, which is limiting growth. Where growth is limited by oxygen, it would be predicted that ester synthesis would be promoted since less acetyl-CoA would be used for biomass formation, and therefore more would be available for esterifcation. However, where another nutrient restricts growth, such as amino nitrogen, lack of oxo-acid skeletons may reduce ester formation.

6.4.1.3 Wort dissolved oxygen concentration. Oxygen is required by yeast during fermentation for the synthesis of sterols and unsaturated fatty acids, both of which are essential for membrane function (see Section 3.5). In controlled fermentations, the initial concentration limits the extent of yeast growth. Dilution of the sterol and unsaturated fatty acid pools between pitched cells and progeny, during growth under anaerobic conditions, produces the requirement for oxygen when the cropped yeast is re-pitched into a fresh batch of wort.

Reportedly, brewing yeast strains can be classified into groups on the basis of a minimum oxygen concentration that produces satisfactory fermentation performance (Kirsop, 1974; Jacobsen & Thorne, 1980). In concentration terms the oxygen requirement ranges from approximately 4ppm to more than 35ppm dissolved oxygen. It is received wisdom that ale strains have a higher oxygen requirement than lager strains; however, this supposition has not been subject to exhaustive testing. The evaluation requires use of pitching yeast with 'anaerobic' physiology followed by assessment of performance of multiple fermentations in separate batches of wort containing a range of initial oxygen concentrations. This is rarely done other than in academic studies.

At production scale, it is more usual to select oxygen concentration based on empirical observation. Often this means using the maximum concentration that can be achieved with the oxygenation system at hand, since this generally ensures a rapid fermentation. In traditional fermentations, which use air as the source of oxygen, this is acceptable. However, in the case of large-capacity vessels, which use pure oxygen, this approach is not good practice. There is an understandable tendency to over-oxygenate worts because it avoids the problems of sluggish fermentation rates and poor yeast growth. However, if too much oxygen is used there is a risk of loss of fermentation efficiency. Thus, for any combination of yeast and wort, a progressive increase in the initial concentration of oxygen results in a concomitant increase in fermentation rate, until a plateau region is reached where rate becomes independent of oxygen concentration. However, the increase in oxygen concentration is also accompanied by a progressive increase in yeast growth as the additional available oxygen promotes biomass formation. This shift in the pattern of carbon assimilation is balanced by a reduction in the yield of ethanol (Fig. 6.22).

The inflection point where fermentation rate and oxygen concentration become independent of each other, varies with yeast strain. It is related to the oxygen requirement group to which the strain belongs. If the yeast is a member of the highest oxygen-requiring group, the inflection point may never be reached.

Since oxygen availability and yeast growth are related, it follows that the formation of yeast metabolites related to yeast growth would also be affected. In particular, beer ester levels decrease in inverse proportion to the increase in yeast growth extent.

As discussed in Section 6.4.1.2, the effects of oxygen on fermentation rate, yeast growth, ethanol yield and formation of beer flavour components related to yeast growth are also modulated by the pitching rate. The patterns shown in Fig. 6.22 would all have been subtly shifted if a different pitching rate had been used. The key parameter is the ratio of oxygen supplied per yeast cell since it is this that controls the quantity of sterol formed per cell. At commercial scale, the time taken to fill vessels and in consequence the time during which yeast cells are exposed to oxygen is also influential, as discussed in Section 6.1.5. There is also the possibility of variable

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Oxygen concentration (ppm)

Fig. 6.22 Effect of varying initial worty oxygen concentration on yeast growth, ethanol and fermentation rate in stirred laboratory fermentations using high-gravity (1060) wort. The pitching rate was 15 x 10s viable cells ml-1 and the temperature was maintained at 11°C throughout the fermentations. Varying oxygen concentrations were obtained as described in Bamforth et a!., 1988).

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Oxygen concentration (ppm)

Fig. 6.22 Effect of varying initial worty oxygen concentration on yeast growth, ethanol and fermentation rate in stirred laboratory fermentations using high-gravity (1060) wort. The pitching rate was 15 x 10s viable cells ml-1 and the temperature was maintained at 11°C throughout the fermentations. Varying oxygen concentrations were obtained as described in Bamforth et a!., 1988).

pitching yeast physiology, which produces an altered requirement for wort oxygenation. Exposure of pitching yeast to oxygen during storage can permit limited sterol synthesis. A further possibility is that should the fermentation conditions be such that oxygen is not the limiting factor, it follows that the cropped yeast will not be entirely depleted in sterol. In both of these scenarios, there will be a reduced requirement for oxygen in the next fermentation to produce optimum performance. To maintain control of fermentation this should be corrected for, as discussed in Section 6.4.2.

6.4.1.4 Pressure. During fermentation, pressure effects may be manifest in three ways. All have the potential to influence performance. First, there is a hydrostatic pressure due to the height of the fermenting vessel. Second, the yeast cells are subject to an osmotic pressure, or variable water activity, which is related to the composition of the wort. Third, in closed vessels it is possible to restrict the outflow of carbon dioxide and allow the fermenter to pressurise. Extremes of pressure have the potential to exert deleterious effects on yeast cells. However, fermentations may be conducted under moderate top pressure with no effect to yeast. Indeed, application of pressure during fermentation is used as a strategy for manipulating ester formation (see Section 3.7.3).

The magnitude of osmotic pressure is a function of wort concentration. The osmotic pressures in concentrated worts can be considerable. Thus, Owades (1981) reported osmotic pressures equivalent to 40 atmospheres (4 x 106 Pa) in some high-gravity worts. Gervais et al. (1992) reported that yeast cells were able to withstand osmotic pressures up to 108 Pa, provided the cells were in an appropriate physiological condition. However, damage could occur if there was a rapid shift from low to high osmotic pressure.

Osmotic effects on yeast cells have a pressure component and a water activity component. Thus, in a solution, the concentration of solids and availability of water (water activity aw) are inversely related. All organisms are capable of survival and/or growth within a given range of water activities but comparatively few can withstand very low values (Hocking, 1988). In the case of brewery wort, this parameter is one of several factors that limit the maximum gravity that can be used without compromising both beer quality and yeast condition (see Section 2.5).

Increased hydrostatic pressure has manifold effects on microbial cells. In yeast it causes ultrastructural changes that result in leakage between intracellular compartments (Shimada et al., 1993). Disruption of many cellular processes due to high pressure have been reported and indeed it is used as a method of sterilisation (Kamihira et al., 1987). The deleterious effects on structure and function of cells have been likened to the effects of high temperature and oxidative stress (Iwahashi et al., 1995). However, these effects occur at comparatively high pressures, typically greater than 100 MPa.

In brewery fermenters, a hydrostatic pressure is generated by the liquid height in the vessel. In this respect, vessel capacity, geometry and the degree of agitation are important. Clearly, in tall vessels, the effect will be magnified. During primary fermentation, agitation is efficient, and yeast cells will circulate continuously throughout the vessel. Consequently, they will be subjected to a constantly changing pressure environment. Undoubtedly this has effects on yeast metabolism and perhaps contributes to the differences in beer flavour when similar worts are fermented in vessels of different aspect ratio.

Top-pressurisation of vessels involves two inseparable components. First, the effects of elevated pressure itself, and, second, those due to the accompanying increased concentration of dissolved carbon dioxide. The effects of both of these components on ethanol fermentation have been studied in the context of fuel alcohol production (Thibault et al., 1987; L'ltalien et al., 1989). Here the impetus was the possibility of using super critical carbon dioxide for in situ recovery of ethanol. These authors reported that hyperbaric conditions (7 MPa) inhibited ethanol formation. The inhibition was reversible by reducing the pressure. Nevertheless, even at these high pressures, it was possible to maintain an ethanol productivity of 10.9gl :h more than would be possible in brewery fermentation. Inhibition by carbon dioxide appears to be more significant than effects simply due to pressure.

Top pressurisation of brewery fermentations is used as a means of modulating yeast growth and the concentrations of beer flavour components where alteration of other control parameters has produced an imbalance. Smaller pressures are used than those described above. In general, the treatment is used to reduce the extent of yeast growth and the concentration of volatile beer components. However, conflicting reports have appeared in the literature as to the precise effects. Rice et al. (1976) described the effects of carbon dioxide top pressure of 22.7 psig (0.16 MPa) on 100 litre lager fermentations. The extent of yeast growth and concentrations of beer volatiles produced at 22°C were the same as those observed at 15°C with no top pressure. The increased pressure had no effect on fermentation rate. Other small-scale trials (Arcay-Ledezma & Slaughter, 1984) reported that pressurisation at 2 atmospheres (2 x 105 Pa) reduced fermentation rate, yeast growth extent and higher alcohols. In addition, at the end of fermentation, levels of vicinal diketones were elevated. In this case a top-fermenting ale yeast was used.

At production scale, pressure fermentation has been used successfully to compensate for the effects on beer flavour of elevated temperature (Nielsen et al., 1986,1987). In this case, a pressure of 1.2 atmospheres (0.12 MPa) was used. The pressure was applied gradually during fermentation. This reduced yeast growth and volatile beer components compared to unpressurised fermentations. Both esters and higher alcohols were influenced but to varying degrees and with no clear pattern. An alternative treatment reported by the same authors and claimed to be as effective as pressurisation in producing the effects described above, was to carbonate the wort. In this case wort was collected with 8 ppm oxygen and carbonated to 0.3%.

Miedener (1978) described fermentations in which increased pressure was used to reduce the concentrations of higher alcohols performed at elevated temperatures. The fermentation was allowed to proceed at atmospheric pressure until approximately 50% attenuation was achieved. At this point the release of C02 was restricted and a top-pressure of roughly 1.8 bar (1.8 x 105 Pa) was allowed to build up. This author suggested that a suitable value for the top pressure was equal to a tenth of the fermentation temperature in degrees Celsius.

Miedener (1978) reported a number of analytical differences between beers from normal and pressurised fermentations. The pH of the latter was higher, possibly a consequence of increased yeast shock excretion of nucleotides and amino acids in response to the higher pressure (see Section 3.7.6). Head retention values were also lower. This was ascribed to an increased content of short-chain (C6-Ci0) fatty acids. Possibly, this suggests that the pressure caused some yeast autolysis. In flavour terms, there was no significant alteration in the concentrations of higher alcohols, except for 2-phenylethanol. In the pressure fermented beer this increased by a remarkable twofold. Ethyl acetate and iso-amyl acetate concentrations were not significantly different but the ethyl esters of hexanoic, octanoic and decanoic acids were approximately doubled in the pressure beers. Both dimethyl sulphide and total vicinal diketones were reduced in the pressure-fermented beers.

Posada (1978) described the effects of pressurisation in spheroconical fermenting vessels (see Section 5.4.3.3). It was confirmed that pressure caused a reduction of yeast growth extent. However, this report made the point that the modulating effects of pressure on the formation of some beer flavour components is yeast strain specific. For example, with two different yeast strains, elevated pressure produced opposite changes in the concentration of 2-phenylethanol. Similarly, the concentrations of other flavour components could be shifted upwards or downwards, by pressure, dependent on the yeast strain. Presumably, these strain dependent variations are the source of the sometimes conflicting reports of the effects of pressure fermentation on beer flavour components.

6.4.2 Automatic control regimes

It may be appreciated that the combination of the effects of the variables which contribute to the initial conditions established at the start of fermentation are complex and interdependent. It is necessary to choose appropriate values for each user variable, and most often this is achieved by a combination of trial and error tempered by previous experience. Unfortunately, even when the most stringent control is applied to each variable, not all inconsistencies in fermentation performance will be eliminated. This is a consequence of variability in the composition of the wort and the physiological condition of the pitching yeast.

By the time the wort has been collected into fermenter there is little opportunity for modifications to be made other than those discussed in Sections 6.1.3 and 6.2. At present, therefore, there is no mechanism for correcting for variability of wort composition. Several approaches for the rapid assessment of the physiological condition of pitching yeast have been proposed (see Section 7.4.2). These so-called 'yeast vitality' tests may be viewed as an extension to the conventional viability test. The aim is to produce information regarding the viable fraction of yeast that is predictive of subsequent performance in fermenter. Results from such tests may be used to form a judgement of fitness to pitch for a particular batch of yeast. Alternatively, the results of the test may be used to calculate the pitching rate that will produce the desired fermentation performance.

The logical development of vitality testing is that assessment of yeast condition should be carried out in fermenter as part of an automatic interactive feed-back control system. Such an approach opens the possibility of correcting for variability in both yeast condition and wort composition.

6.4.2.1 Economics. The greater the sophistication of the fermentation control system, the more the potential cost of installation and operation. Therefore, before installing such systems it is essential to assess the potential economic benefit and compare this with expenditure. This is a difficult calculation since the real cost penalty of variable product quality is not easy to quantify. Certainly, the costs of sophisticated on-line monitoring and control devices can be justified only where several large capacity fermenting vessels are used. In breweries that are more traditional, it should be sufficient to ensure adequate fermentation control by careful manual regulation of the pertinent parameters.

The potential savings to be made by ensuring rigorous fermentation control may be illustrated in the following example. Assume a brewery with 20 x 1500 hi vessels in which fermentations with optimal control take 10 days to complete. Allowing 3 days for filling, cooling, emptying and cleaning, the potential annual productivity is:

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