Maintenance of stocks of pure yeast strains in the laboratory is discussed in Section 7.1. Here are described the methods in which these stocks are used to introduce new yeast cultures into the brewery.
All fermentations generate yeast sufficient to re-pitch two or more further fermentations. In some breweries, particularly traditional ale top-cropping types, this cycle proceeds ad infinitum and single yeast cultures have been in use for many years. Frequently, these may be mixed cultures; however, fermentation performance is considered to meet the needs of the process without the need for propagation of fresh yeast. More commonly, however, and without exception in modern breweries, new cultures of yeast are introduced periodically to replace existing stocks.
This is prudent for a number of reasons. Most importantly, it provides an opportunity to introduce a culture of known and guaranteed identity. Continued serial fermentation and cropping carries with it the risk that variants may be selected for within the yeast population. Consequently, over a number of generations of fermentations a gradual drift may occur in the properties of the yeast (see Section 188.8.131.52). This is particularly the case where the cropping regime may inadvertently select for variants. For example, early cropping from the cones of cylindroconical fermenters may select for flocculent sub-populations within the yeast. It is also reported that the formation of petite mutants (see Section 184.108.40.206), which are known to perform poorly in fermentation, is the most common spontaneous mutation in brewing yeast strains (Ernandes et al., 1993). Periodic re-introduction of a new yeast line taken from a master culture limits the opportunities for the occurrence of this source of variation.
Even in the best-managed brewery, the production environment provides opportunities for the introduction of contaminants to bulk yeast. These may be in the form of bacteria (Section 8.1.2) or wild yeast (Section 8.1.3). The consequences of contamination with wild yeast can be significant, resulting in process changes (floccu-lation, fining) or flavour changes (phenolic, medicinal character). With respect to bacteria, contamination with Obesumbacterium spp. (Kara et al., 1987b) can result in the formation of carcinogenic nitrosamines from nitrite (see Section 220.127.116.11). From another standpoint, cross-contamination of yeast strains may occur where several are in use in a single brewery.
Continued serial re-pitching of yeast may be associated with gradual deterioration in yeast condition, which can result in a decline in fermentation performance. This is unlikely in the case of rapid top-cropped fermentations where there is an opportunity to ensure that the fraction of yeast retained is that which is produced when fermentation is at its most vigorous. In addition, such cropping regimes are to some extent self-purifying (see Section 6.7.1). In the case of bottom-cropped fermenters, particularly large-volume cylindroconicals, there is opportunity for high levels of contamination of yeast with trub. The possibility of selecting for non-standard yeast variants has been alluded to already. The use of very large vessels and the tendency towards high-gravity brewing has undoubtedly increased the stresses to which production yeast is subject. It has been demonstrated that senescence of yeast cells (see Section 18.104.22.168) may be associated with declining performance of yeast since some cropping regimes may select for larger and therefore possibly older cells within yeast populations (Barker & Smart, 1996; Smart & Whisker, 1996; Deans et al., 1997).
The frequency of introduction of newly propagated yeast into the brewery is a decision for the individual brewery since there are no immutable rules. A typical regime in a modern brewery built and operated to high standards of hygiene would be to introduce propagated yeast every 15-20 generations. However, some breweries would consider this excessive and only allow 5-10 generations to elapse before introducing new yeast. Two contrary points of view may be considered in this respect. First, a fermentation management decision may have been taken within a particular brewery, which makes it mandatory that new yeast cultures are introduced after a given number of generations. Typically in this scenario a number of different yeast lines, each of varying generational age, would be in use at any given time. Introduction of a new yeast line would be phased to replace a line that has reached the end of its operational lifetime. This method totally disregards the fermentation performance of that particular yeast line.
On the other hand, if a particular yeast line is performing satisfactorily and is microbiologically 'clean', there will be a natural desire to continue brewing with such yeast, even though it may have completed its allotted number of generations. This highlights the need for methods of testing the yeasts' physiological condition, which are predictive of subsequent fermentation performance. In other words the so- called 'vitality tests' described in Section 7.4.2.
Continuing to use yeast that is performing in a satisfactory manner has other advantages. There is an economic cost to propagation. Apart from the capital investment, the revenue costs are obviously proportional to the frequency of use. In addition, it is commonly observed that the first generation fermentation using newly propagated yeast is atypical. In consequence, the first generation beer has to be blended. Occasionally, the non-standard behaviour may be extended over the first few fermentations. Clearly, this would tend to mitigate against frequent propagation.
The reasons for non-ideal behaviour in first fermentations are obscure. Hammond and Wenn (1985) reported the experiences of a brewery where one newly propagated yeast strain produced slow fermentation performance for the first few generations. This was shown to be due to impaired ability of the strain to utilise maltotriose. The causes of the defect were not elucidated and the effect was somewhat transitory, disappearing after 7-10 generations. Since this effect was seen only with a single yeast strain it suggests that the problem was not due to propagation per se, but was a feature of the particular yeast. Several reports have described yeast undergoing abrupt changes in flocculence and there is evidence to suggest that this is due to an inherent genetic instability (see Section 22.214.171.124). Altered flocculence and defects in sugar assimilation may be related. Thus yeast that is not capable of utilising maltotriose would stop growing and flocculate before the cells comprising the normal population. Such variant cells could be selected for in cone cropping of fermenters. If such genetic variants can arise in fermenter, it follows that the same phenomenon could occur in propagator. It also follows that should such variants arise it would not be a reflection of propagator performance.
In fact, it can be demonstrated that, provided the design and operation of the propagation plant is adequate, there is no fundamental reason why first-generation propagations should be different from later ones. It may also be surmised that the common contention that newly propagated yeast gives less than ideal performance is a consequence of poor propagator design and operation. In consequence, firstgeneration fermentation pitching rates are often inadequate and the yeast is in a stressed condition.
The requirements of a propagator are summarised as follows:
(1) Hygiene is of prime importance and the design and operation of the propagation plant must ensure that a pure yeast culture is generated. Since the propagator is to supply yeast for brewing it is essential that it is not a source of contamination. This is an obvious requirement of propagation but one that is not always adhered to.
(2) The terminal cell count must be adequate to achieve the desired pitching rate in the first generation fermentation.
(4) The physiological condition of the yeast must be consistent and appropriate for subsequent fermentation.
(5) The cycle time of propagation should be as rapid as possible, both for economy and to minimise the risk of contamination, and should use the fewest possible number of vessels.
(6) Terminal cell counts from the final propagation stage should be as high as possible so as to allow high step-up ratios and minimise the effects on the first generation fermentation of the 'barm ale' introduced with the propagated yeast.
The use of pure culture plant in brewing is, of course, not new and the first yeast propagators were introduced by Hansen in 1883 (Curtis, 1971). The process, therefore, has a long history and several distinct systems have been developed. Nevertheless, all propagation regimes basically consist of a sequence of yeast cultures of progressively increasing volume, starting in the laboratory and culminating in a terminal stage which contains sufficient yeast to pitch the first production scale fermentation. Variations on this theme are possible, such as semi-continuous systems, which maintain cultures at the small brewery scale and thereby reduce the requirement for repeated laboratory propagation.
126.96.36.199 Laboratory propagation. The aim of the laboratory phase of fermentation is to generate a pure yeast culture of sufficient size to provide an adequate pitching rate for the first stages of brewery propagation. The terminal laboratory culture must be held within a container, which will allow transfer to the brewery under conditions of asepsis, and there be transferred into the brewery propagation vessel under aseptic conditions.
Laboratory propagation uses standard microbiological apparatus. It must be performed to the highest possible standards using skilled personnel. Initial stages may use artificial media such as yeast extract, peptone, glucose. Wort may be used for the terminal laboratory stage; however, it must be sterilised by autoclaving prior to use. A typical laboratory propagation regime is shown in Fig. 7.3. The scheme shown is a suggestion only and several variations are possible. It is sensible to limit as far as possible the number of aseptic transfers, since these represent the points of greatest risk of contamination. In general, a volume scale-up factor of about 1:10 is satisfactory.
The terminal laboratory stage requires a purpose built piece of apparatus, such as
24 h static culture at 25° C
2 x 10 ml in universal bottle
3 days at 25°C on orbital incubator
2 x 100 ml in 250 ml conical flask
3 days at 25°C with constant stirring and aeration through sterile filter and glass sinter
3 litre culture in 10 litre aspirator
3 days incubation at 25°C with constant aeration and stirring
20 litre terminal culture
Fig. 7.3 Schematic flow diagram for laboratory propagation of brewing yeast.
that shown in Fig. 7.4 and described by Boulton and Quain (1999). This consists of a heavy gauge stainless steel flask with a capacity of approximately 25 litres fitted with a number of ports passing through the top-plate assembly. The latter is removable for cleaning purposes. Before use the flask is filled with brewery wort and the flask and contents sterilised by autoclaving. If experience shows it to be necessary, antifoam may be added to the wort before sterilisation. After cooling and prior to inoculation, the wort should be aerated by sparging for at least 30 minutes with air or pure oxygen. Sterility is maintained by passing the gas through a microbiological quality gas filter. High rates of oxygen transfer are facilitated by passing the inflowing gas through a stainless steel candle. Inoculation is via a specific port that terminates in a male fitting, which is wrapped to maintain sterility. Immediately before inoculation the male fitting is joined, using appropriate aseptic precautions, to a matching female fitting, also wrapped, attached to the side-arm of the aspirator used for the 3 litre culture stage (Fig. 7.4). The inoculum culture is transferred by gravity after opening the relevant valves.
After inoculation, the flask is aerated continually with air or pure oxygen. Oxygen transfer rates are further improved by constant agitation using a powerful magnetic stirrer and a follower in the flask. The exhaust gas is vented to atmosphere via another microbiological grade filter. If the gas flow rates are high it is advisable to locate a water condenser between the gas outlet port and the sterile filter. Aseptic sampling is possible via a tube, that extends to near to the bottom of the flask. The sample may be withdrawn by temporarily restricting the outlet gas line.
It is convenient to mount the flask in a purpose-built trolley to facilitate transport. The trolley can be designed to hold the magnetic stirrer and a gas cylinder to provide
^^ brewery seed vessel Sample point
^^ brewery seed vessel Sample point
motive power during transfer of the culture from flask to brewery seed vessel. When the culture is ready the flask is disconnected from the gas inlet supply and transferred to the brewery. Connection to the brewery seed vessel is via a dedicated line, which terminates in a sterile wrapped fitting, which is designed to attach securely to the inlet point on the seed vessel. During transfer the culture should be agitated continuously using the magnetic stirrer. Transfer of the culture is achieved by applying top pressure via the gas exhaust line.
188.8.131.52 Brewery propagation. The raison d'être of production scale propagation plant is to provide conditions that favour yeast growth. It follows that the same conditions will also favour the growth of contaminants and, therefore, good hygienic design and operation is absolutely essential. Vessels are fabricated from stainless steel with particular attention being paid to interior finish and fittings to facilitate cleaning. Inlet and outlet gas lines are via microbiological grade steam-sterilisable filters. After cleaning, vessels are sterilised with steam. In operation, vessels require attemperation by the application of cooling. A means of introducing sterile air is provided. Traditionally, propagators are not usually agitated. Sample points must be of the steam sterilisable type. This is especially important in the case of seed vessels where the sample point is often used for introducing the laboratory inoculum. As a further precaution against the possibility of infection, it is prudent to operate the vessels under 0.5-1.0 bar top pressure.
The microbiological sensitivity of the process requires that facilities are provided for wort sterilisation. Frequently, this is achieved by provision of a separate sterile wort holding tank from which wort is taken to feed the propagation vessels. Wort may be sterilised, in situ, in the holding tank by application of steam to external jackets. Alternatively, the wort may be sterilised by passage through a heat exchanger during filling. In the absence of a holding tank the wort may be sterilised in the propagation vessels themselves.
To assist with hygiene it is advantageous to site propagation plant in a separate room. This should have all-sealed surfaces to facilitate cleanliness, have self-closing doors and preferably be operated under a positive air pressure. Pipework and valves connecting propagation vessels and the rest of the brewery must be designed to the highest hygienic standards. Apart from cleaning pipework in between transfers there must be a means of sterilising (see Section 8.2.1).
Brewery propagators vary greatly in their sophistication, capacity and yield. As in the case of fermenter design, there are traditional and modern propagators which although basically similar, perhaps have a different underlying design philosophy. Thus, all propagators are built to high hygienic standards, to guard against the risks of contamination (see Fig. 7.5). Certainly, newer installations are likely to be superior in this respect than their older counterparts. However, there is a difference in that traditional systems are frequently operated not to obtain the maximum yeast count within a minimum time but rather to generate yeast. Many consider it important that this yeast is in a physiological state which resembles, as nearly as possible, that of
conventional pitching yeast. This is supposedly achieved by careful control of temperature and oxygenation. Conversely, more modern installations are generally designed and operated with thoughts of maximum yield and shortest cycle times uppermost.
In the brewery, yeast is propagated using sterile wort. Therefore, assuming no supplements are made to the wort, the only means of regulating yeast growth and extent is by manipulation of temperature and oxygenation. All brewing yeast strains have optimal growth temperatures of around 30°C (see Section 4.2.2). Propagation at this temperature would obviously be very rapid; however, this potential benefit is rarely taken advantage of. In traditional systems, relatively low temperatures are used, typically up to 20°C for ale yeasts but lower for lager strains. Commonly a relatively high temperature may be used in the first vessel followed by a gradual reduction in temperature at each subsequent stage with the terminal propagation being performed at the same temperature as the first fermentation (Maule, 1979).
An excess of oxygen during fermentation promotes yeast growth and the same is true in the case of propagation. This strategy also is not adopted with traditional (and many modern) propagators. Usually more oxygen is provided than would be the case for fermentation - typically, intermittent aeration throughout the entire propagation. However, aeration rates are typically low and the absence of mechanical agitation ensures that oxygen transfer rates are generally poor. In this sense, many propagation vessels are little more than hygienically designed fermenters!
The reasons for propagating at relatively low temperatures and limiting the availability of oxygen are two-fold. First, there is a assumption that these conditions will produce yeast with physiology similar to pitching yeast. In addition, this ensures that the yeast suffers no thermal shock. It is difficult to reconcile these assumptions on purely scientific grounds. Second, regard must be made to the 'beer' which will be pitched with the culture yeast. High propagation temperatures and excess oxygen favour elevated levels of higher alcohols and acetaldehyde and reduced esters (see Chapter 3). Clearly where the step-up ratio is small, as is frequently the case with traditional propagators, there is a risk of adverse flavour effects in the first generation beer due to carry-over of non-standard beer. Of course, where propagators are designed to achieve only modest cell yields, and therefore only small scale-up factors can be employed, the problems of non-standard barm ale are to some extent a self-fulfilling prophecy.
Traditional propagation systems use modest scale-up factors between each stage, typically 1 to 5. This can produce a very cumbersome process when reasonably large fermenters require to be serviced. For example, Maule (1979) described a system, illustrated in Fig. 7.6, used in a lager brewery to propagate sufficient yeast for 800 hi fermenters. As may be seen, the entire propagation process from laboratory to first fermentation is lengthy, taking a matter of weeks to complete. A short cut, which circumvents the initial laboratory stages, is to recycle part of an intermediate propagation to provide a new inoculum, as shown in Fig. 7.5. This approach, often termed 'intermittent propagation', produces a much shorter cycle time but does not address one of the prime reasons for propagation, namely the need to guard against selection of non-standard variants. In addition, it is not a suitable system for a brewery that requires to propagate several yeast strains.
Terminal laboratory phase
1st propagation vessel
1st propagation vessel
2nd propagation vessel
3rd propagation vessel
Fig. 7.6 Scheme for brewery yeast propagation (from Maule, 1979).
The trend towards very large capacity fermentation vessels places great demands on propagation plant. The system described in Fig. 7.6 is capable of providing yeast for an 800 hi fermenter. However, cylindroconicals of twice this capacity or greater are common. Frequently, these large fermenters have been installed within a brewery with an existing propagation plant built to service smaller vessels. In this case, it is common practice to use smaller fermenters, if available, for the first generation fermentations to generate sufficient yeast to pitch larger vessels. Another variation on this theme is to part-fill large fermenters in order to achieve the correct pitching rate using an inadequate propagation plant.
Clearly, strategies such as part filling fermenters is at best a poor compromise and does not make best use of fermenter capacity. Furthermore, such a system becomes very unwieldy in breweries using several yeast strains. For example, a brewery that uses six yeast strains, for a maximum of ten generations, would require propagation plant to be in operation on a permanent basis to avoid the possibility of having no yeast to pitch. An alternative strategy is to use propagators that are capable of delivering high yields within a short process time. Several systems of this type have been designed, which share in common the use of relatively high growth temperatures and aerobic conditions (Geiger, 1993; Schmidt, 1994, 1995; Brandl, 1996; Ashurst, 1990; Von Nida, 1997; Boulton & Quain, 1999; Westner, 1999).
Typically, brewing yeast growing on high-gravity (15-18°Plato) wort under continuously aerobic conditions and temperature within the range 25 to 28°C would yield a terminal yeast count of 200-300 x 106 cells ml 1 within 24-36 hours. Providing the process is terminated when the maximum cell count is achieved and aeration is discontinued, there is no opportunity for diauxy (the transition between fermentative and oxidative physiology) to occur. Consequently, yeast will be in a catabolite repressed condition, essentially similar to conventional pitching yeast. It is, of course,
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