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Fig. 6.3 Effect of wort specific gravity and temperature on the solubility of oxygen (♦, 10°C; 15°C; A 20°C) with (a) air-saturated wort at NTP and (b) oxygen-saturated wort at NTP.

T is the temperature at NTP;

P02 is the partial pressure of oxygen in the gas stream; C is the desired oxygen concentration (mgkg *); Fw is the wort flow rate (kg s *).

Where pure oxygen is used, P02 equals the hydrostatic pressure within the wort main. Where air is used the value of P02 reduces to the value of the hydrostatic wort main pressure multiplied by the factor 0.2094.

In practice, it may be difficult to ensure perfect solution of the inflowing gas. Better control may be achieved by locating a dissolved oxygen meter up-stream from the gas addition point. Using a suitable controller, the rate of gas addition may be regulated automatically to give a desired dissolved oxygen concentration. It should be noted that the dissolved oxygen probe must be located at a point before yeast addition in order to avoid errors of underestimation due to yeast oxygen uptake.

The problems of inefficient oxygen solution in wort mains have been addressed by a novel approach developed at the BRi (Rennie & Wilson, 1975; Wilson, 1975). With this technique, the wort stream is blended with water, which has been supersaturated with oxygen by a combination of chilling and high pressure. Thus, it was claimed that at 2°C and 10 bar g a dissolved oxygen concentration of 700 mgl 1 could be achieved. It was suggested that by maintaining a suitable back-pressure, the oxygenated water and wort could be blended with no gas break-out. Although ingenious, this approach has not been adopted, probably because of the complexities that it would add to a conventional oxygenation system. Thus, the requirement to prepare and hold the water feed, which, of course, would have to be sterile. Furthermore, there would be the need to prepare wort at an altered gravity and temperature to correct for the effects of blending, and the requirement for an accurate blending system.

Although dissolved oxygen meters provide a direct measure of the concentration of oxygen dissolved in wort, their application to controlling the process does have some disadvantages. The requirement to locate the probe at a point up-stream of yeast addition has been mentioned already. This can be a problem if there is insufficient length of main between the points of oxygen injection and measurement such that there has not been time for complete solution to occur. Many meters have slow response times, which can lead to inaccuracies, and they require frequent maintenance.

In sophisticated oxygenation systems (Fig. 6.4) it is usual to control gas addition using a thermal mass flow meter. These devices allow addition of a calculated total mass of oxygen to a known volume of wort. The required mass of oxygen may be metered in throughout the entire wort run. However, it is more convenient to arrange to add all the oxygen with the first 90% of the wort volume and none with the last 10%. This guards against the possibility of wort addition being completed before all the oxygen has been added. Dissolved oxygen measurement is not required although an in-line probe may be provided as a precautionary measure such that wort flow is suspended if the flow of oxygen fails. Output from the meter can be fed to a datalogger and provide a permanent record of oxygen addition.

Thermal mass flow meters allow precise control of addition of oxygen; however, it

Pressure regulator Low pressure Mass flow

Pressure regulator Low pressure Mass flow

Glycol Water

Fig. 6.4 System for wort oxygenation.

Glycol Water

Fig. 6.4 System for wort oxygenation.

is still necessary to ensure complete solution. Stainless steel sinters with a large surface area and pore size of approximately 20 (im, used in combination with an in-line mixer and pressurisation of the wort main, provide a suitable method. Although pressurisation of the wort main aids oxygen solution, some gas break-out in the fermenter, which will be at atmospheric pressure, is inevitable. Furthermore, it is not possible to measure the dissolved oxygen concentration in fermenter since by the time wort collection is completed, the yeast will also be present and assimilation of oxygen will have commenced.

In practice, it is usual to arrive at an optimal oxygen dosage rate based on empirical observation of fermentation performance. It follows that the most important consideration is to use a system that provides a consistent wort dissolved oxygen concentration. It is important to ensure that all potential sources of wort oxygenation are eliminated other than that added deliberately. If dilution water is added after wort, it should be either deaerated or included in the calculation of oxygen added. Post-pitching air rousing in fermenter, which is sometimes used as a method for ensuring proper dispersal of yeast, is not recommended where precise control of wort oxygen concentration is considered to be critical.

The practice of adding oxygen over the whole period of wort collection has been questioned. Lodolo et al. (1999) reported that the optimum time for oxygen addition was four hours after pitching. The authors claimed that this allowed the dissolved oxygen tension to be reduced from 16mgl 1 to 8mgl 1 with no alteration in fermentation performance. For brewing at production scale, it was recommended that all the yeast was pitched with the first batch of anaerobic wort. Oxygen was added with subsequent batches of wort.

6.1.3 Control of extract in fermenter

At the point in the brewing process when fermenting vessels are filled, there is little opportunity to exert control over wort composition other than regulation of the dissolved oxygen concentration, as described already. Some additions may be made, in fermenter, to modify parameters such as total fermentability, sugar spectrum, metal ion and mineral salt content (see Section 6.2). However, the gross wort composition is established during its preparation.

It is necessary to control the total extract added to the fermenter. This parameter is the product of the specific gravity of the wort and its total volume, often expressed in litre degrees. For reasons of efficiency of vessel utilisation, it is important that fermenters are operated at their maximum working volumes. In other words they should be filled to a level where there is just sufficient freeboard to allow retention of foam. In addition, because of its impact on both beer quality and yield, the specific gravity of the wort must be controlled to achieve an initial desired value. However, it is on the basis of total extract that the yeast pitching rate and wort dissolved oxygen concentration should be based. In practice, this is problematic because both yeast and oxygen are added to the wort before collection is complete and the final value for total extract may not be known with great precision.

How are these difficulties reconciled? In fact, with a greater or lesser degree of success depending on how carefully the process is managed. The total extract delivered to the fermenter is controlled by operations within the brewhouse, notably, the quantities of raw materials used and the manner of their treatment during conversion to clarified and cooled wort. In a typical modern brewery, the value for total extract may be measured at the whirlpool stage. Based on this measurement the total yeast to be pitched and mass of oxygen to be added should be calculated. This is rarely done and it is assumed that operations in the brewhouse are sufficiently controlled to produce a consistent batch of wort. Yeast pitching and oxygenation rates are usually then based on this nominal value.

In some breweries, the wort may be held in a receiving vessel located between brewhouse and fermenter. At this stage, adjustments to gravity and clarity may be made and the total extract measured, thereby allowing an accurate calculation of the quantity of yeast to be pitched and oxygen to be added. Unfortunately, this method is extravagant in the use of expensive vessels, slows process times and presents potential risks for microbial contamination. It is of most use where fermenters may be filled with a single batch of wort, which is not the case in many modern breweries. For these reasons, it is more common to fill fermenters directly from the whirlpool.

The quantity of extract transferred to fermenter is dictated by the efficiency of whirlpool operation. Very large capacity fermenting vessels require several individual batches of wort to be discharged from the whirlpool and this magnifies the effects of losses of extract. Wort delivered from the brewhouse is concentrated and needs to be diluted to achieve a desired gravity. This may be performed in fermenter by off-line measurement of gravity and subsequent dilution. If this is approach is used, it is essential to make sure that the contents of the vessel are mixed to homogeneity. This may be achieved by gas rousing, pumping wort through an external loop to promote mixing or use of a mechanical stirrer. The latter option is preferable since it is efficient and carries only a small risk of disturbing the wort oxygen concentration. External loop systems do not provide good mixing if dilution water has been added in such a way that there has been appreciable layering. Gas rousing is the least satisfactory method of mixing the contents of fermenters. If an inert gas is used such as nitrogen or carbon dioxide, some dissolved oxygen will be lost due to gas stripping. Conversely, if air or oxygen is used, control of wort oxygen concentration is lost. In deep vessels, dilution water should be added from the bottom since the density differential will promote mixing. The dilution water should be deaerated to avoid the inadvertent and uncontrolled addition of oxygen.

Wort dilution may be performed in-line during collection using an automatic blending system. This approach probably offers the best chance of maintaining rigorous control of the collection process. Degrees of automation are possible. The simplest system is that in which the volume and specific gravity of the wort are measured in the whirlpool. With knowledge of the desired final gravity, the quantity of dilution water required may be calculated and blended, in-line with the wort, as it is delivered to the fermenter. The blending system should preferably be a proportioning system, which adds the water at a constant ratio throughout the entire run. This provides wort of a consistent gravity and temperature and therefore, constant oxygen solubility. Automatic in-line or in-tank measurement of specific gravity is possible (see Section 6.3.2.3). Such sensors are suitable for automatic control of wort dilution. Systems such as this have been developed; however, they are used for dilution of high gravity beers after fermentation, or possibly monitoring of fermentation progress and not for control of wort collection.

The final stage of wort collection is to provide a record of the total extract in the fermenter. This requires a measure of the specific gravity and the volume. In some countries such as the United Kingdom and Belgium, which used to levy excise duties based on wort sugar content, there was a statutory requirement to maintain an accurate record of wort volume and concentration. In consequence, all fermenting vessels were gauged to allow volume assessment via dip measurements. Since by the time wort collection was completed, the yeast would have been added and fermentation commenced, it was not possible to measure the initial specific gravity. Instead, this was inferred from a value termed the original gravity (OG) of the wort. This used a correction factor for the quantity of fermentable sugar used in yeast metabolism but not converted to ethanol (see Section 1.4). Although excise payment is now based on ethanol concentration in beer, records of OG and wort volume are still maintained to allow assessment of fermentation efficiency.

The change in excise law to end-product duty allows greater latitude in the methods used to assess total extract. For example, vessel volumes may be measured using intank level sensors. Daoud (1991) describes the application of ultrasound to level measurement. Here a transducer in the base of the vessel senses liquid depth by the time taken to detect the return of a pulsed signal reflected back from the gas-liquid interface. Depth measurement must take into account possible interfering factors. For example, differential volume changes in liquid and vessel due to variations in temperature, and the need for the liquid to be absolutely still. This avoids errors due to swirl and the effects on volume of entrained gas. An alternative method of wort volume measurement is to use in-line flow meters to record the total volume of liquid added to fermenter. Such devices may be needed as part of the control systems for yeast pitching and wort oxygenation.

6.1.4 Control of yeast pitching rate

The pitching rate is defined as the concentration of yeast suspended in wort at the start of fermentation. The process of inoculating wort with yeast is described as 'pitching' and the yeast reserved for this purpose 'pitching yeast' (for a review see O'Connor-Cox, 1998b). The term, as the word suggests, derives from the physical act of throwing yeast into the fermenting vessel. For example, in Scandinavian countries it was custom to prepare a circular structure made from many wooden laths woven together and called a pitching wreath. This was recovered from a previous fermentation. The large number of crevices and surfaces in the wreath provided places for yeast cells to lodge and survive the period of storage between fermentations. These provided the inoculum, when the wreath was pitched into a subsequent fermentation. In the United States the term 'brink' yeast is synonymous with pitching, deriving in this case more obviously from that which is present at the start of the fermentation.

The aim of the process is to obtain a defined suspended yeast count in wort at the start of fermentation. In fact, this parameter is rarely measured since by the time wort collection is completed the yeast may have started to multiply, thereby negating the value of measurements. Instead, it is usual to rely on a related parameter such as addition of a known wet weight of yeast, or volume of yeast slurry. Control of pitching rate has two components. First, an analysis of the yeast reserved for pitching, and, second, a method for accurately transferring a desired quantity of this yeast into the wort. Several approaches have been developed to accomplish these two tasks. These are associated with varying degrees of precision and each has advantages and disadvantages.

6.1.4.1 Direct weight of yeast cake. In some traditional fermentations, particularly using top-cropping ale types, it is common practice to store the skimmed yeast in the form of pressed cake. In subsequent fermentations, the pitching rate is controlled simply by addition of a defined weight of yeast cake. The pressed yeast may be added directly to the fermenter although it is more usual to re-suspend it in cold water in a separate pitching tank to facilitate subsequent dispersion in the wort.

The quantity of yeast used is based on an assumption that there is a defined correlation between yeast wet weight and cell count. Thus, the UK brewers' 'rule of thumb' that 1 lb pressed yeast per UK barrel is equivalent to 10 x 106 cells per ml in the pitched wort. In fact, this relationship is obviously an approximation, there being some variation depending on yeast cell size. However, this method provides reasonably precise control of pitching rate since yeast skimmed from top-cropping fermenters tends to be relatively free from contaminating non-yeast wort solids, the major potential source of error. It does have the disadvantage that the yeast is inevitably exposed to considerable atmospheric oxygen during handling. This is especially the case where slurried cake yeast is pitched into empty open square fermenters before the wort is pumped in.

From a fermentation control standpoint, exposure of pitching yeast to oxygen is undesirable since some limited sterol synthesis may occur. This promotes excessive yeast growth in the subsequent fermentation if the wort oxygen concentration is not reduced (see Section 6.4.1). This is a small disadvantage in the case of typically small-scale ale fermentations, which use this pitching rate control system. Thus, maintenance of high process efficiency through controlled yeast growth is less important than providing conditions that ensure a rapid onset of fermentation and a profuse crop. Exposure of pitching yeast to air and oxygenation of wort promotes both of these.

6.1.4.2 Metered addition of yeast slurry. Pitching yeast is most commonly stored in the form of a slurry in which the cells are suspended in beer derived from the previous fermentation. The pitching rate of a subsequent fermentation is controlled by the metered addition of a known weight or volume of slurry, either directly to the empty fermenting vessel or more usually in-line with the wort during collection. Analysis of the pitching yeast slurry is required in order to make the calculation of the quantity of yeast to be added. In the majority of breweries, the analysis is performed in the laboratory on samples removed from yeast storage vessels.

Two approaches to the analysis are possible. First, determination of the proportion of suspended solids, expressed as percent weight to weight or weight to volume. The relative proportions of yeast and beer in a sample of slurry are determined by cen-trifugation and direct weighing of pellet and barm ale. Occasionally, the slurry may be analysed using a graduated centrifuge tube and the solids content defined as percent suspended solids volume to volume. In the interests of precision, this latter approach cannot be recommended. On a separate portion of the sample, the yeast viability may be determined using a microscopic counting and dye staining method, such as the methylene blue test (see Section 7.4.1). This allows a correction factor to be introduced so that pitching rates are controlled in terms of viable spun solids. Such tests are valuable aids in that they also allow assessment of pitching yeast quality or condition (see Sections 7.4.1 et seq.). Thus, it would be usual to reject pitching yeast with viability less than a minimum quality limit.

The second method of yeast slurry analysis is to determine the cell count per unit mass or volume of slurry. This has the benefit that unlike the simpler centrifugation procedure there is an opportunity to correct for the presence of non-yeast solids. Furthermore, It allows pitching rates to be controlled directly in terms of yeast count. Two methods of cell counting are commonly used.

First, using an electronic particle counter, which gives a rapid and automatic measure of the suspended yeast cell count. Second, direct microscopic enumeration using a counting chamber such as the haemocytometer (EBC Analytica Micro-biologica II, Methods 3.1.1.1 and 3.1.1.2, respectively).

Both counting methods have advantages and disadvantages. Electronic counting is rapid and simple but can usually cope only with cells which are borne singly, and errors may accrue due to the presence of non-yeast solids. Some of the more sophisticated devices are capable of discriminating between particles of varying sizes and indeed will produce a size distribution curve. However, yeast cells in floes or chains may still be scored as single cells and the presence of non-yeast solids with a particle size similar to that of yeast will introduce further errors of under-estimation.

Prior to counting highly flocculent yeast it is necessary to disrupt cell clumps with an ultrasonic treatment or use of a chemical deflocculent such as maltose. These treatments tend to be of limited efficacy with some yeast strains. Electronic counters give no information regarding yeast viability and a separate analysis is required to make correction for this parameter.

Direct counting with a haemocytometer requires no sophisticated apparatus other than a suitable microscope, and used in conjunction with a vital stain it allows simultaneous assessment of total and viable yeast count. It is possible to discriminate between yeast cells and non-yeast particles and chain-forming strains may be counted without problem. As with the electronic approach, large yeast floes must be disrupted before enumeration is possible. The major drawback with direct microscopic counting is that it requires trained personnel. Even in the hands of skilled operators, large errors in repeatability and precision are the norm. Siebert and Wisk (1984) compared both microscopic and electronic yeast counting and concluded that there was a nine-fold difference in precision in favour of the latter method. Furthermore, electronic counting was easier, less tiring to operators and more rapid. However, correction for yeast viability was not considered in this study.

Miller et al. (1978) suggested that the total viable fraction of pitching yeast slurries could be determined accurately and speedily by measurement of ATP concentration using firefly bioluminescence (see Section 8.3.4.1). The authors reported that the ATP content of yeast cells, in any given physiological state, is relatively constant. Since it is absent from dead cells and non-yeast solids, the concentration of this metabolite in pitching yeast slurries showed a positive correlation with viable yeast concentration. A method was developed in which yeast slurry was sampled from storage vessels and analysed photometrically. The results allowed computation of an optimum pitching rate, which in production trials was shown to be 2.0 (ig ATP per ml wort. When used at production scale, this method was shown to produce consistent fermentation performance. The method has obvious attractions, but unfortunately yeast slurries require to be diluted some 100-fold to obtain a result. This implies that careful and skilled handling of samples is required in order to avoid what could be potentially very large experimental errors.

With knowledge of the yeast concentration, it is possible to calculate the quantity of slurry to be added to fermenter to achieve a desired pitching rate. This may be a manual operation, in which the required quantity of slurry is pumped from storage vessel to fermenter. Alternatively, automatic systems use a controller which receives output from load cells fitted to the pitching yeast storage vessels or an in-line flow meter. When the appropriate quantity of slurry has been transferred directly to fermenter, or injected into the wort main during collection, the controller terminates the process by switching off the pitching pump and closing the pathway between storage vessel and fermenter.

6.1.4.3 Cone to cone pitching. In some breweries that use cylindroconical fermenting vessels it is practice to pitch with yeast taken from the cone crop of one fermentation and transferred directly into the empty cone of a new fermentation. This procedure avoids the need for intermediate storage tanks. The yeast should be in good condition since there has been no opportunity for deterioration due to prolonged storage. Similarly, there should be no exposure to atmospheric oxygen which could influence the requirement for subsequent wort oxygenation. On the other hand, it is an inflexible system since dedicated storage tanks free fermenting vessels to be filled or emptied as dictated by the requirements of production.

As with conventional pitching systems, this method also requires removal of a yeast sample from fermenter for off-line analysis so that the quantity of slurry to be transferred can be assessed. This is a highly inaccurate procedure. Yeast crops in the cones of cylindroconical fermenters are heterogeneous due to layering of yeast cells and non-yeast solids (Boulton & Clutterbuck, 1993). Furthermore, stratification of the yeast crop in terms of cell size/age is also possible (see Section 6.7.2). In consequence, analysis of small off-line samples does not provide a representative assessment of the total yeast crop. Furthermore, removal of yeast from fermenter cones may be associated with complex mixing effects due to vessel geometry. This can cause a central plug of yeast to exit first, followed by the portion of the crop nearest to the walls. It is, therefore, difficult to control accurately the transfer of a known quantity of viable yeast from one fermenter to another.

A method has been described which reportedly surmounts some of these problems (Anonymous, 1992). This approach employs in-line sensing devices, which, by monitoring flow rate and density, provide a continuous measure of the proportion of solids suspended in the yeast crop as it is transferred from cone to cone. Used in conjunction with a batching device, the system claimed to provide accurate control of pitching rate when in routine use at production scale.

6.1.4.4 Use of near infra-red turbidometry. Quantification of suspended cell counts, or other particles, indirectly by turbidometry is a well tested method. It relies on there being a correlation between cell concentration and light scattering, usually measured at a particular wavelength. Turbidometric sensors have been designed for use in the quantification of yeast cell concentration for the control of yeast pitching rate (Riess, 1986). This approach has the major advantage that in-line measurements can be made and, therefore, a fully automated pitching rate control system is possible (Fig. 6.5). Yeast concentration is quantified by measurement of turbidity in response to incident radiation in the near infra-red range, which is claimed to give the best linear relationship. Two NIR sensors are used. The first is located in the wort main, up-stream of the yeast injection point and provides a measure of the turbidity due to the unpitched wort. The second sensor is placed down-stream of the yeast injection point and an in-line mixer, but before the oxygen injection point, provides a measure of the turbidity due to the pitched wort. A back-pressure orifice valve located between the control and motoring apparatus prevents gas breakout which would interfere with readings. In operation the difference in turbidity reading between the two sensors is maintained at a set-point, by modulation of the yeast slurry injection control valve, thereby maintaining a constant yeast count within the pitched wort.

The value of the set-point is derived empirically for individual combinations of yeast strain and wort quality. The operating range of the detection system is 1 x 106 to 50 x 106 cells ml l. This has implications regarding the manner in which the pitching system must be used. Where yeast is pitched in-line with the wort it is best practice to add the slurry as quickly as possible. This ensures that the fermentation

Yeast in

Oxygen in

Yeast injection valve

Oxygen Injection valve

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