7.1.1 Maintenance of stock cultures
The storage and assured supply of brewing yeast strains is an important step in the propagation cycle. Although arguably obvious, it is perhaps not generally appreciated that storage and supply are critical to the ongoing success and consistency of yeast management in the brewery. Indeed as noted by Guldfeldt and Piper (1999), 'despite the fundamental importance of a reliable supply of pure and stable cultures, culture maintenance is often afforded low priority'. This attitude is unfortunate and is inappropriate. Correct and robust procedures for culture maintenance (and supply) are not a 'nice to have' but a 'must have'! To this end, long-term yeast storage should meet four basic criteria (Quain, 1995). Ideally, the method should be genuinely long term in that yeast can be (i) stored for many years without (ii) compromising viability or (iii) genetic stability. Moreover, the method should (iv) be accessible and as technically straightforward as possible. This section identifies the popular options for yeast storage and critically assesses their success in terms of yeast survival (short and long term), genetic stability and simplicity. The strengths and weaknesses of these approaches are summarised diagrammatically in Table 7.1 and reviewed in practical detail by Kirsop (1991).
Table 7.1 Critical comparison of methods for long-term storage of yeast (after Quain, 1995). Method Survival Shelflife Genetic stability Simplicity
Subculture on agar
Freezing in liquid nitrogen
Performance is graded as: superior, (H) intermediate and @ poor.
126.96.36.199 Third-party storage. For simplicity, third-party storage cannot be beaten! The responsibility for secure maintenance and storage is handed over to a culture collection that periodically supplies the strains back to the depositor. This option is clearly attractive to small breweries who wish to periodically propagate their strains but do not wish to establish the necessary laboratory facilities and expertise for internal maintenance and storage. Such facilities are offered by commercial culture collections such as the Alfred Jorgensen Laboratory (www.ajl.dk) in Denmark who supply yeast cultures to more than 100 breweries worldwide. Another approach is take advantage of the secure 'confidential safe deposit' service offered by culture collections such as the National Collection of Yeast Cultures (NCYC) in the UK and the BCCM in Belgium (for details see Section 188.8.131.52). In the case of the NCYC, although marketed as a back-up in the event of catastrophe, the service satisfies the needs of 'third-party' storage of brewing yeasts for periodic propagation. Here, at reasonable cost, strains are preserved by freeze-drying (Section 184.108.40.206) and in liquid nitrogen (Section 220.127.116.11). Two cultures of the strain are supplied annually to the depositor with the option of further cultures charged at the collection's prevailing rates. Although such an approach lacks true flexibility it meets the objectives of simplicity and secure, assured storage. For breweries wishing to store more than one or two strains, it is likely that such an approach would become, over time, prohibitively expensive.
18.104.22.168 Storage by subculturing. Subculturing micro-organisms by streaking out on solid agar plates (or slopes) or by inoculation in a liquid broth are fundamental practical tools of microbiology and, by definition, require technical resource and facilities. However, as noted by Kirsop (1991), subculturing is 'simple to do, quick to carry out, and relatively inexpensive'. Unfortunately, the simplicity of subculturing is its undoing! Although universally used across microbiology, it is generally recognised that repeated subculturing of micro-organisms (and subsequent storage at < 4°C) is a less than ideal protocol for long-term storage. Further, in the case of brewing yeast, viability cannot be assured by storage (at < 4°C) for longer than 4-6 months. Consequently, the need for periodic subculturing can be labour intensive, particularly with large collections of yeast strains. These considerations are further exacerbated by concerns that these approaches result in 'strain drift' through the formation and enrichment of genetic and phenotypic variants (Wellman & Stewart, 1973). These have been reported for flocculation (Russell & Stewart, 1981; Kirsop, 1991; see Section 4.4.4), respiratory deficient petites (Russell & Stewart, 1981; see Section 22.214.171.124) and, generally, for morphological and physiological properties (Kirsop, 1974). A further practical concern that can be levelled at serial subculturing is that poor technique and cross-contamination can compromise strain identity and purity. Given these many concerns, it is no surprise that subculturing cannot be recommended as an approach to long-term storage of brewing yeasts.
126.96.36.199 Storage by drying. Although as described by Kirsop (1991), drying protocols include niche methods such as the 'paper replica method' and the 'silica gel method', the approach described here is restricted to the more popular 'freeze-drying' method.
Lyophilisation or freeze-drying of brewing yeasts has long been practised (Kirsop, 1955) and today remains a popular approach to the long-term storage of yeast and other micro-organisms. Freeze-drying involves water removal by sublimation from the frozen sample using a centrifugal dryer. It is very much a small-scale procedure with typically 100 (il of yeast suspension (at 106ml *) and equivalent volume of suspending 'protectant' being dried and sealed under vacuum in a glass ampoule. Its success is built around the method's relative simplicity (for details see Kirsop, 1974,
1991), low running costs (although start-up can be appreciable) and, perhaps most significantly, the ease of handling, storage and transport when dried. However, from experience, the end user can occasionally experience frustration and difficulty in scoring and breaking glass ampoules.
Although it continues to be used by culture collections such as the NCYC and National Collection for Marine and Industrial Bacteria (NCIMB), freeze-drying has - in recent years - had a bad press. Most damning of all is the general acceptance that post freeze-drying the viability of the culture is usually low. Early observations (Kirsop, 1955) at the then Brewing Industry Research Foundation (now Brewing Research International) reported the average viability for 29 strains of S. cerevisiae to be 4.6% two days after drying. Nine months later, the average viability was effectively unchanged at 4.3%. Similar conclusions were made by the same worker (Kirsop, 1991) some 36 years later which showed that at the NCYC the average viability for freeze-dried cultures of the genus Saccharomyces was 5%. This, if anything, is higher than other yeast genera in the NCYC! Such poor performance appears to be due to the formation of intracellular ice during the freezing stage of the process. In one report (Berny & Hennebert, 1991), the viability post-freeze-drying of a strain of S. cerevisiae increased from 30 to 98% by controlled slow freezing (3°C min *) in the presence of skimmed milk and other protectants. However work in the 'related world' of active dry bakers' yeast suggests that dehydration of the yeast below 15% moisture is the trigger for loss in viability (Bayrock & Ingledew, 1997). A report by Russell and Stewart (1981) found freeze-drying to be the least successful protocol for storage of brewing yeasts, being outperformed by traditional subculturing methods and, most dramatically, by storage in liquid nitrogen. In addition to a catastrophic loss of viability (five of eleven brewing strains were totally dead!), changes in flocculation and increased frequency of petite mutation were seen in some strains. By way of explanation, freeze-drying has been reported to impact at the level of the yeast genome by causing chromosomal breaks (Barros Lopes et al., 1996; see Section 188.8.131.52).
Guldfeldt and Piper (1999) have described an alternative, simpler but lengthy approach to preparing dried yeast used by the Alfred Jorgensen Laboratory to supply pure yeast strains. Yeast cells are suspended in sorbitol (20% v/v), incubated for six hours, filtered under vacuum and then dried over calcium chloride for up to 12 days at 10°C. Although technically much simpler than the standard freeze-drying method, the Guldfeldt and Piper approach suffers similarly when post-storage viability is compared to storage in liquid nitrogen.
Ironically, outside of the rarefied world of culture collections, dried yeast has long found commercial application in the wine and baking industries. Until recently, compared to dried wine yeasts, dried brewing yeasts have fared poorly because of low and inconsistent viability (O'Connor-Cox & Ingledew, 1990). However, lager yeast strains have now been successfully dried using a combination of fed batch propagation and fluidised bed drying - a process similar to that used in the production of active dry bakers' yeast. Although viability post drying is 50-60%, it has been shown with two lager strains to be maintained at this level for up to 12 months at 4°C (Muller et al., 1997) or 10°C (Fels et al., 1999). Although originally marketed for use in primary fermentation (Muller et al., 1997), production scale trials in Israel and
Tanzania (Fels et al., 1999) and in Belgium (Debourg & van Nedervelde, 1999) have focused on the role of dried yeast in yeast propagation. Although fermentation performance is typical of the 'control' wet yeast, there is a need for a more exhaustive analysis of genetic and physiological stability. It remains to be seen whether this approach can go some way to resolving the technical issues surrounding freeze-drying or even be scaled down to achieve similar flexibility.
184.108.40.206 Storage by freezing in liquid nitrogen. Storage in liquid nitrogen is the crème de la crème of cell storage methods. It is used to 'successfully store fungi, bacteriophage, viruses, algae, protozoa, bacteria, yeasts, animal and plant cells and tissue cultures' (Snell, 1991). For yeast, cryogenic storage in liquid nitrogen at its boiling point (— 196°C) has long been recognised as the best approach to maintaining viability and integrity. Its application to brewing yeast has been described at Labatts (Wellman & Stewart, 1973; Russell & Stewart, 1981), the UK's NCYC (Kirsop, 1991), Bass (Quain, 1995) and Scottish Courage (Jones, 1997). If performed correctly, storage in liquid nitrogen has no impact on cell viability over periods of seven months (Guldfeldt & Piper, 1999), two years (Russell & Stewart, 1981), 26 months (Kirsop & Henry, 1984), three years (Wellman & Stewart, 1973) or eight years (Kirsop, 1991). Similarly, the general view is that storage at — 196°C does not trigger any genetic changes. Despite this, Wellman and Stuart (1973) reported changes for one lager strain in flocculation and an increase in respiratory mutants. However, subsequent comparative work by the group (Russell & Stewart, 1981) gave liquid nitrogen storage a clean bill of health.
To be successful, storage in liquid nitrogen requires adherence to basic protocols (Kirsop & Henry, 1984; Kirsop, 1991). Cells cannot simply be plunged into liquid nitrogen! Prior to freezing, yeast cells are grown oxidatively (Quain, 1995), an approach which has been shown to best afford protection against the potentially lethal stresses of freezing and thawing (Lewis et al., 1993). Cells are then suspended (at c. 100 x 106ml *) in fresh media containing a cryoprotecterant (typically glycerol at 2.5-5% v/v). Subsequent freezing is a two step process: (i) the temperature is progressively reduced over two hours from room temperature to — 30°C followed (ii) by immersion in liquid nitrogen at — 196°C. The rate of cooling in the initial step is critically important to the success of cryogenic storage, as it is during this phase that the cells are dehydrated. Over time the fraction of water (in the suspending media) which is frozen increases whilst, in turn, the concentration of salts in the unfrozen fraction rises. This progressive increase in osmotic pressure results in the yeast cells losing water and shrinking with a decrease in surface area and increase in the thickness of the cell wall. It is considered that the rate of shrinkage is a vital element in preventing cell damage through intracellular ice formation (Kirsop, 1991). Indeed a strong correlation has been shown between the incidence of intracellular ice and the loss of yeast viability (Morris et al., 1998). Conversely, revival following storage at — 196°C is performed rapidly by immersion in water at 25 to 37°C.
The use of liquid nitrogen lends itself to the storage of collections of microorganisms. In our experience (Quain, 1995), a substantial collection of current production strains (c. 25) is maintained alongside (in a separate vessel) a collection (c. 50) of'historical' brewing strains and other yeasts of interest. Typically (Kirsop & Henry, 1984), yeast suspensions are stored in ampoules (0.5 ml) or, preferably in short coloured straws (0.2 ml) derived from children's drinking straws (Fig. 7.1). This later small-scale approach is more flexible, enabling large numbers of straws to be prepared and stored. Perhaps the one disadvantage of such permanent storage is recovery. As noted in Section 7.1.2, this is not quick. Indeed, the ideal and flexible solution is a hybrid of long-term storage in liquid nitrogen coupled with short-term storage (< 6 months) on agar slopes or plates at 4°C.
In keeping with the scale of cryopreservation, the capital cost of cryovessels (Fig. 7.2) and ancillary equipment can be significant. Further, liquid nitrogen must be safely contained and 'handled' with appropriate safety procedures and precautions. However, tellingly, the need for maintenance is low and is limited to periodic (but regular) topping-up with liquid nitrogen from a 'buffer' tank. This is important as storage in the nitrogen vapour phase (—139°C) reportedly (Kirsop, 1991) allows some biochemical and biophysical processes to continue. Accordingly, similar reservations apply to storage of yeasts at — 70°C which, in our experience, also results in significant losses in viability (Wendy Box, unpublished observations).
7.1.2 Deposit, recovery and validation of identity
As described in Section 7.1.1, long-term storage of yeast strains via serial culture on agar plates or slopes cannot be recommended. This is unfortunate, as this mode of storage is convenient and readily available for sub-culturing at the outset of propagation. Comparatively, storage regimes such as freeze-drying and freezing in liquid nitrogen are inflexible inasmuch that the culture must be recovered, resuscitated and 'grown-up' prior to use. Undoubtedly such constraints have limited the widespread use of liquid nitrogen and, to a lesser extent freeze-drying.
Whatever the method, the process significance together with the demands of good laboratory practice, demand some degree of checking of yeast quality both pre- and post-storage. Although perhaps taken as 'a given', it is disappointing that the literature is, with one exception (Quain, 1995), bereft of detailed comment on how such checking may be performed.
Bass Brewers' approach to yeast deposit and supply was described in detail at the EBC in Brussels (Quain, 1995). Here, storage of production yeast in liquid nitrogen is a central service consisting of conventional and 'new' microbiological tests. The package is controlled and documented to meet international quality standards (ISO 9002). The driver for such complexity and control is simple! Yeast is critically important to product quality, consistency and diversity so assurance of the right yeast strain of the right physiological and microbiological quality is paramount. Consequently as noted by Quain (1995), 'there is no room for error in yeast supply'. Risks of contamination or strain mix-up must either be removed or minimised and controlled by applying best operating practices together with appropriate monitoring.
Risks are minimised by a number of basic rules. Irrespective of the number of strains being handled, all activities within 'yeast supply' require two people. This is deemed necessary to negate the risk of operator fatigue, to provide technical support and to observe/confirm actions. Further, only one strain is handled at a time. Total assurance of yeast strain identity and purity are achieved - both going in and coming out of liquid nitrogen - by the use of a variety of microbiological plate tests (Table 7.2, see Section 8.3.3) and RFLP-based DNA fingerprinting (see Section 220.127.116.11). To verify performance - and to instil confidence in the results - each medium is challenged with control micro-organisms that will or will not grow (Table 7.2). Similarly, on deposit into liquid nitrogen strains are identified 'blind' by DNA fingerprinting.
Although originally three times a year (Quain, 1995), yeast supply to Bass Brewers
Table 7.2 Microbiological QA of yeast supply (after Quain, 1995).
Selective media Solid media Aerobic incubation +ve control — ve control
Selective media Solid media Aerobic incubation +ve control — ve control
Table 7.2 Microbiological QA of yeast supply (after Quain, 1995).
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